A di-glycine motif in the cytoplasmic tail of CD3ε required for transmission of allosteric changes in the TCR and full CD3ζ phosphorylation

A di-glycine motif in the cytoplasmic tail of CD3ε required for transmission of allosteric changes in the TCR and full CD3ζ phosphorylation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A di-glycine motif in the cytoplasmic tail of CD3ε required for transmission of allosteric changes in the TCR and full CD3ζ phosphorylation Rut Tercero, Elena R. Bovolenta, Nadia Martín-Blanco, Alice Lombard, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4594242/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract How the T cell receptor (TCR) recognizes its antigen ligand and transmits this information to the cytoplasm for T cell activation is still a matter of debate. A large body of experimental data suggests that the TCR undergoes allosteric changes upon ligand binding that are responsible for the outside-in transfer of information. One of the hallmarks of TCR allostery is the induced exposure of a proline-rich sequence (PRS) in the cytoplasmic tail of the CD3ε subunit, enabling binding to the cytoplasmic adaptor protein Nck. We show here that a glycine-to-alanine mutation (G169A) in a conserved di-glycine motif upstream of the PRS impairs TCR binding to Nck upon stimulation with an activating antibody. Furthermore, the mutation hinders CD3ε tyrosine phosphorylation and, interestingly, selectively affects CD3ζ subunit phosphorylation at Tyr83. In addition, the G169A mutation impairs the phosphorylation of Lck at Ser59. Taken together, these data support the concept of TCR allostery and highlight the existence of a regulated sequence of interactions between TCR subunits and associated effector proteins. Biological sciences/Immunology/Signal transduction Biological sciences/Immunology/Lymphocytes/T cells/T cell receptor T cell receptor Allostery CD3ε Nck binding Lck recruitment CD3ζ phosphorylation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION The T cell antigen receptor (TCR) is responsible for recognizing antigens and can be considered the foundation of the adaptive immune system due to the ability of T cells to orchestrate the type and intensity of the immune response to pathogen-encoded antigens. In mammals, the majority of T cells (αβ T cells) recognize antigens in the form of peptides inserted into a groove formed by molecules of the major histocompatibility complex (MHC). αβ T cells bearing a TCR capable of recognizing the complex of the antigenic peptide and an MHC allele (pMHC complex) with sufficient affinity are activated and enter a program of proliferation and differentiation. This process generates various effector and memory T cells that are responsible for mounting an immune response to eliminate the antigen and retaining antigen memory for faster and stronger responses to future encounters with the same antigen. The TCR is a multi-subunit complex consisting of the TCRα and TCRβ subunits, which are responsible for recognizing pMHC through their variable sequence regions, and the invariable CD3γ, CD3δ, CD3ε, and CD3ζ (also known as CD247) subunits. The TCRα and TCRβ subunits have very short cytoplasmic tails and are, in principle, incapable of interacting with cytoplasmic effector proteins. In contrast, CD3 subunits have longer cytoplasmic tails that have been shown to recruit effector proteins such as the tyrosine kinase Lck, the tyrosine kinase ZAP70, and the adapter protein Nck upon pMHC binding to the TCRα/β subunits. Somehow, the information about pMHC binding must flow from the TCRα/β subunits to the cytoplasmic tails of the CD3 subunits. The most obvious mechanism would be that of allosteric changes resulting from conformational changes that transfer outside-in information about pMHC binding from the TCRα/β ectodomains to the CD3 ectodomains and transmembrane regions and from these to the cytoplasmic tails. Some structural data have been generated supporting the existence of ligand-induced conformational changes in the ectodomains of the TCR 1 – 4 . However, the strongest evidence to date for conformational changes in the TCR has come from the observation that TCR triggering by pMHC or agonistic anti-CD3 antibodies induces the exposure of various motifs in the cytoplasmic tails. The earliest evidence was the finding that a proline-rich sequence (PRS) unique to the tail of CD3ε does not adopt a conformation prone to bind the N-terminal SH3 domain (SH3.1) of Nck when the TCR is at rest 5 . Ligation of the TCRα/β or CD3 ectodomains with agonistic antibodies or binding of cognate pMHC results in the PRS adopting a conformation able to bind the SH3.1 domain of Nck 5 , 6 . These results suggest that there must be an outside-in mechanism for transmitting allosteric changes to the PRS of CD3ε. The CD3γ, CD3δ, and CD3ε subunits are located within a 30 Mb region on human chromosome 11 (and on chromosome 9 in mice), with CD3γ and CD3δ positioned head-to-head and separated by 1.6 kb 7 . These CD3γ and CD3δ subunits result from a relatively recent duplication, as only mammals have two distinct genes for them, while birds and lower vertebrates (reptiles, amphibians, and fish) possess only one gene encoding a glycoprotein with intermediate homology to CD3γ and CD3δ8. CD3ε is already present in cartilaginous fish and has remained highly conserved. This conservation likely reflects its role in forming dimers with both CD3γ and CD3δ in mammals, which are at least partially redundant. The cytoplasmic tails of the CD3 subunits contain either one (CD3γ, CD3δ, and CD3ε) or three (CD3ζ) tyrosine- and leucine-based motifs with the consensus sequence YxxL/I (x)6–9 YxxL/I. These motifs, known as ITAMs (Immunoreceptor Tyrosine-based Activation Motifs), are rapidly phosphorylated upon TCR activation by the src family tyrosine kinases Lck and Fyn 9 . When ITAMs are phosphorylated at both tyrosines, they become high-affinity (nM) docking sites for proteins with two SH2 domains in tandem, such as the Syk family tyrosine kinases, Syk and ZAP70 10,11 . The presence of multiple ITAMs in a single TCR complex (up to 10, considering one CD3ζ homodimer and two CD3ε subunits) has sparked debate over whether this multiplicity serves purely to amplify signals or also has qualitative effects. The hypothesis that each ITAM can bind different sets of signaling proteins is supported by the fact that the amino acids around the two YxxL/I motifs and those in the spacer between them differ for each ITAM but have been conserved throughout vertebrate evolution. Interestingly, a comparison of the amino acid sequences of the cytoplasmic tail of CD3ε in vertebrates shows that the most highly conserved feature is not the ITAM but the PRS 12 . Other highly conserved amino acids have also been identified. In this work, we made point mutations in several evolutively conserved amino acids in the CD3ε cytoplasmic tail and identified Gly169 as an important amino acid for TCR signaling. Its mutation alters the earliest detectable activation events, including induced exposure of the PRS, phosphorylation of some ITAMs in CD3ζ, and recruitment of Lck to the TCR. We discuss the relevance of these findings to our understanding of how the TCR transduces activation signals. RESULTS An evolutionarily conserved glycine motif located between the Basic-Rich Sequence (BRS) and the Proline-Rich Sequence (PRS) in the cytoplasmic tail of CD3ε is essential for T cell activation induced by the T Cell Receptor (TCR). A comparison of the amino acid sequences in the cytoplasmic tail of CD3ε reveals a remarkable pattern of conservation across ontogeny, spanning from cartilaginous fishes to mammals (Fig. 1 a). The most conserved feature is the Proline-Rich Sequence (PRS, PxxPxPDY), followed by the tyrosine residues of the Immunoreceptor Tyrosine-based Activation Motif (ITAM). Other sequences previously reported to be crucial for outside-in signal transmission within the TCR are also preserved, including the Basic-Rich Sequence (BRS) 13 , 14 and the RK motif involved in recruiting Lck 15 . Additionally, here are other conserved amino acid residues located between the BRS and the PRS. Notably, we noticed the presence of a conserved proline residue (Pro161 in human CD3ε) and one or more glycine residues, forming a di-glycine motif (Gly169-Gly170 in human CD3ε), present in all vertebrates and conserved in mammals (Fig. 1 a). Characterization of the structure of the cytoplasmic tail of murine CD3ε by Nuclear Magnetic Resonance (NMR) (PDB = 2K4F) revealed a random coil throughout the 57 amino acids of the polypeptide, except for two turns of an α-helix around the C-terminal tyrosine of the ITAM, an α-helix turn at the end of the PRS and N-terminal tyrosine of the ITAM, and an α-helix turn immediately after Pro161 (Fig. 1 b). The residue corresponding to Gly169 is located within a long unstructured sequence. To assess whether mutation to alanine of different residues in human CD3ε affected the TCR's ability to activate T cells in response to ligation with an anti-CD3 antibody, we first generated a CRISPR/Cas9 full knockout of CD3ε in the Jurkat T cell line. Subsequently, the resulting CD3εKO cell line was transfected with either wild-type CD3ε or alanine mutants corresponding to residues Arg155 and Arg156 of the BRS, residue Pro161, residues Gly169 and Gly170 of the di-glycine motif, and residues Arg192 and Lys193 of the RK motif. Stable clones were selected, and those exhibiting TCR expression at the cell surface similar to that of wild-type Jurkat cells (Fig. S1 in the Supplementary Information) were chosen for functional analysis. To this end, all Jurkat clones were stimulated with various concentrations of anti-CD3 antibody, and IL-2 present in the supernatant was measured by ELISA. As expected, the CD3εKO line failed to produce IL-2 in response to anti-CD3 stimulation (Fig. 1 c). All mutants significantly reduced IL-2 production compared to the wild-type control. Of note, cells bearing the G169A mutation exhibited the least responsiveness to anti-CD3 stimulation, releasing IL-2 at levels as low as the CD3εKO control (Fig. 1 c). In a separate set of experiments, Jurkat CD3εKO cells were transduced with lentiviral constructs expressing either wild-type CD3ε or the G169A or G170A mutants, and the resulting polyclonal population was stimulated with different doses of anti-CD3. The response to anti-CD3 stimulation was evaluated based on the expression of CD69 and CD25 as activation markers. Cells bearing either glycine mutation showed deficient responses to TCR triggering (Fig. 1 d and 1 e). These results demonstrate that mutation of glycine residues 169 and 170 to alanine in CD3ε diminishes the human TCR's ability to respond to stimulation with an anti-CD3 antibody. The G169A mutation in CD3ε has a differential impact on the phosphorylation of specific tyrosine residues in CD3ζ. To begin dissecting the mechanisms by which mutation of the di-glycine motif impairs TCR-triggered T cell activation, we first investigated some of the earliest events after TCR triggering, namely the tyrosine phosphorylation of the CD3ε and CD3ζ ITAMs. We focused our analysis on the G169A mutation, given that this has a stronger impact on IL-2 production (Fig. 1 c). In anti-CD3 immunoprecipitates, we observed that the phosphorylation of the N-terminal tyrosine (Tyr166) of CD3ε was significantly inhibited by the G169A mutation (Fig. 2 a). While CD3ε has just two phosphorytable tyrosine residues in its single ITAM, CD3ζ contains three ITAMs in its cytoplasmic tail, accounting for a total of six phosphorylatable tyrosines 16 . A diagram illustrating the residue numbers for these six tyrosine residues within the three ITAMs of human CD3ζ is presented in Fig. 2 b. A deficiency in CD3ζ phosphorylation in Jurkat cells expressing the G169A mutant was noticed in Western blots of postnuclear supernatants of whole-cell lysates with a pan-phosphotyrosine-specific antibody (Fig, 2c). In addition to a putative defect on the tyrosine phosphorylation of CD3ζ, a clear defect in the phosphorylation of other proteins was noticed. According to their mobility in SDS-PAGE, those proteins could correspond to phospho-ZAP70 and phospho-Lck (Fig. 2 c). Interestingly, the putative phospho-CD3ζ bands showed distinct high- and low-molecular-weight forms, and the G169A mutant reduced the phosphorylation of the upper form of apparent high molecular weight (Fig. 2 c). To determine if the G169A mutation in CD3ε primarily affected the phosphorylation of CD3ζ to its highest molecular weight form, we examined the effect of the mutations on the tyrosine phosphorylation of CD3ζ using a phospho-specific antibody for Tyr142 (ITAM ζc). Tyrosine-phosphorylated CD3ζ in Jurkat cells with a wild-type TCR appeared as two protein bands of different molecular weight, with the slowest mobility one presumably corresponding to the fully phosphorylated form. While the G169A mutation did not abolish tyrosine phosphorylation of CD3ζ, it did lead to a noticeable reduction in the ratio of highly phosphorylated to low-phosphorylated forms (Fig. 2 d). We further examined the effects of the G169A mutation on CD3ζ phosphorylation using two additional available antibodies specific for Tyr83 (ITAM ζa) and Tyr111 (ITAM ζb) in postnuclear total cell lysates. Western blot analysis with the Tyr111-specific antibody revealed a protein band corresponding to the low-molecular weight form and a faint band corresponding to the high-molecular weight form, although only in WT cells (Fig. 2 e). Intriguingly, western blotting with the Tyr83-specific antibody showed only one protein band corresponding to the high-molecular-weight form in wild-type cells (Fig. 2 f). This single protein band was reduced in Jurkat cells expressing the G169A mutation compared with WT. Altogether, these findings suggest that the low-molecular weight form of phospho-CD3ζ is phosphorylated at Tyr111 and Tyr142 but not at Tyr83, whereas the high-molecular-weight form is phosphorylated at Tyr83, Tyr142, and, to a lesser extent, at Tyr111. Mutation G169A inhibited the phosphorylation of CD3ζ at Tyr83 but not at Tyr111 or Tyr142 (Fig. 2 d-f), indicating that the mutation in CD3ε specifically impairs the phosphorylation of Tyr83 in the first ITAM of CD3ζ. Western blotting with antibody specific for total CD3ζ, phosphorylated and unphosphorylated, served as a loading control and to indicate that the unphosphorylated CD3ζ has the mobility of low apparent molecular weight one (Fig. 2 g). The effect on the phosphorylation of the other three tyrosine residues, for which suitable antibodies were not available, remains unknown. Nevertheless, these results suggest that the G169A mutation has a specific impact on a tyrosine residue located in the upstream, membrane-proximal ITAM of CD3ζ. The G169A mutation impairs proximal TCR signaling and the phosphorylation of Lck at Ser59 by ERK. To investigate the consequences of the G169A mutation on TCR signaling downstream of the TCR, we decided to measure the phosphorylation of the tyrosine kinases Lck and Zap70, which are tightly associated with early signal transmission. A general cartoon depicting the hierarchy of phosphorylation events following TCR activation is shown in Fig. 3 a. The activated TCR becomes phosphorylated on the tyrosine residues of its ITAMs by Lck. Different tyrosine phosphorylation sites have been identified in Lck, with two of the most important being the autophosphorylation site Tyr394 in its catalytic domain, which activates Lck, and the inhibitory site Tyr505 located at the negative regulatory tail 17 . Double-site tyrosine phosphorylation of the ITAMs by Lck creates docking sites for the cytoplasmic tyrosine kinase Zap70, which becomes recruited to the TCR. Once bound to the TCR, Zap70 becomes phosphorylated, including at the activating site Tyr319 18 . Active Zap70 phosphorylates, among other targets, the membrane-bound adapter protein LAT, which serves as a docking site for PLCγ1. Activation of PLCγ1 leads to activation of, among others, the Raf-ERK MAP kinase pathway. Raf activates the dual kinase MEK, which phosphorylates ERK at the Thr202 and Tyr204 sites. Active ERK is part of a feedback loop since it phosphorylates Ser59 in the unique region of Lck 17 . The effect of such phosphorylation on Ser59 is debated, with some data supporting a reduction in Lck activity 19 and others supporting enhanced activity by inhibiting the recruitment of the phosphatase SHP-1 20 . Upon stimulation with anti-CD3 antibody in Jurkat cells bearing the G169A mutation phosphorylation at Tyr319 of Zap70 was strongly inhibited compared to that found in wild-type CD3ε Jurkat cells (Fig. 3 b). Similar results were obtained for phosphorylation of LAT at Tyr132 (Fig. 3 c); phosphorylation of ERK by MEK was inhibited as well (Fig. 3 b). We studied the effect of the G169A mutation on the phosphorylation of Lck at both the activating site Tyr394 and the inhibitory site Tyr505 in total cell lysates. We found that stimulation of both WT and G169A cells with anti-CD3 did not increase the total content of phospho-Lck, regardless of the anti-phosphosite antibody used (Fig. 3 d), consistent with previous findings 21 . However, there was a clear increase in the apparent molecular weight of Lck induced by anti-CD3 stimulation. Specifically, a higher molecular weight band appeared after 5 minutes of stimulation, rather than at the earlier time point of 1 minute, and this band increased in abundance after stimulation at the expense of a low-molecular weight band (Fig. 3 d). The G169A mutation reduced the stimulation-induced increase in the upper high-molecular weight form at the expense of the low-molecular weight form, as visualized by a diminished high/low ratio increase compared to WT cells (Fig. 3 d, below). Since both Lck bands were equally detected by the anti-phosphoTyr394 and the anti-phosphoTyr505 antibodies, we investigated if the differences in both Lck bands could be due to phosphorylation at the Ser59 site. By probing whole cell lysates of WT Jurkat cells with the anti-phospho-Lck(Ser59) antibody, we found that phosphorylation at that site peaked at 5 minutes after stimulation with anti-CD3 antibody (Fig. 3 e). Such induced phosphorylation was blocked in the presence of the MEK inhibitor UO126, thus proving that Ser59 phosphorylation was promoted by ERK when activated by MEK. Interestingly, the mobility of the Lck band detected by the anti-phospho-Lck(Ser59) antibody corresponded to the high-molecular weight form. Consequently, we investigated if the G169A mutation reduced the phosphorylation of Lck at Ser59 compared to WT Jurkat cells and found that this was the case (Fig. 3 f). The same blotting membrane was reprobed with the anti-phosphoTyr394 antibody, which confirmed that the high-molecular weight Lck band corresponded to the form phosphorylated at Ser59 and that such phosphorylation was inhibited in cells bearing the G169A mutation. Together, these results show that the G169A mutation in CD3ε inhibits the activation of early downstream effectors of TCR signaling, including Zap70, LAT, and ERK, and that it has a selective inhibitory effect on the feedback loop of ERK on Lck phosphorylation at Ser59. The G169A mutation impairs the outside-in transmission of conformational changes in the TCR, preventing the exposure of the PRS in CD3ε. To elucidate a possible mechanism for the deficient phosphorylation of specific tyrosine residues in CD3ε and CD3ζ when the TCR bears the G169A mutation, we investigated if this mutation impaired the transmission of the conformational change from the extracellular domains of the TCR to the PRS of CD3ε. To this end, we performed a pull-down assay on detergent cell lysates of Jurkat WT and G169A cells with beads coated with GST protein fused to the SH3.1 domain of Nck1, as schematized in Fig. 4 a. In WT cells, stimulation with anti-CD3 for 1 minute was already sufficient to induce the binding of the triggered TCR to the SH3.1 domain (Fig. 4 b, uncropped blots are in the Supplementary Information). This induced binding was observed by Western blotting with the three antibodies used, specific for CD3ζ, CD3γ, and TCRα, indicating that the entire TCR shifted from a non-competent Resting state to a competent Active state. Compared with a WT TCR, the G169A mutation impaired the anti-CD3-induced binding of the TCR to the SH3.1 domain (Fig. 4 b and 4 c), suggesting that the G169A mutation impaired the transmission of the conformational change along the cytoplasmic tail of CD3ε. Despite the inducible binding of the entire TCR to GST-SH3.1, some binding of CD3ζ, but not of the other subunits, was detected at time 0 (Fig. 4 b). In another GST-SH3.1 pull-down experiment, the blotting membrane was incubated with the anti-phospho-CD3ζ(Tyr142) antibody. This showed some binding to the beads of the low-molecular weight form of CD3ζ with low phosphorylation even in lysates from resting cells (Fig. 4 d). However, the binding of the high-molecular weight form of CD3ζ was exclusively detected after stimulation for one minute or longer, suggesting that the active TCR binding to the SH3.1 domain is fully tyrosine phosphorylated. The G169A mutation impaired such induced binding of the high-molecular weight, fully phosphorylated, form of CD3ζ (Fig. 4 d) and suggests that adoption of the Active conformation by the TCR, including the cytoplasmic tail of CD3ε, is required for full phosphorylation of CD3ζ. DISCUSSION Unlike membrane tyrosine kinase receptors, the TCR has no intrinsic enzymatic activity and relies on the recruitment of cytoplasmic kinases that act in a concerted manner to phosphorylate tyrosine residues in the cytoplasmic tails of TCR subunits, and these phosphorylated tyrosines become docking sites for the recruitment of effector proteins. Even with this simplistic interpretation of the earliest mechanisms of TCR signaling, the TCR does not appear to be a "passive" receptor that is phosphorylated and dephosphorylated in response to what other membrane and cytoplasmic proteins determine. One of the arguments that the TCR is not just an on/off switch is the diversity of signaling subunits, cytoplasmic tails and ITAMs 22 . The cytoplasmic tails of the CD3γ, CD3δ, CD3ε and CD3ζ subunits have been conserved since the emergence of the first jawed vertebrates, and this conservation does not only concern the tyrosine residues conforming to the ITAMs, but also extends upstream, downstream and to the inter-tyrosine sequences. A comparison of all the sequences of the cytoplasmic tails of CD3ε revealed that the most conserved motif was the PRS, which is involved in the recruitment of Nck, followed by the central tyrosine residues of the ITAM and other motifs previously described: the BRS 13 and the RK motifs 13 , 15 . In this paper, we have made point mutations affecting conserved residues of these novel motifs and found that all of them impaired the signaling capacity of the TCR, resulting in the release of IL-2. We focused our attention on an uncharacterized di-glycine motif located between the BRS and the PRS. A conservative mutation of one of these residues, Gly169, to alanine, resulted in the greatest reduction in IL-2 release, as well as reductions in the expression of the activation markers CD69 and CD25. As to what might be the root cause of the signaling defect in the TCR, we have found that the G169A mutation impairs the induced exposure of the PRS for Nck binding, suggesting that Gly169, and probably Gly170, are required for the outside-in transfer of the anti-CD3-induced conformational change in the TCR all the way from the transmembrane domain to the PRS. It has been proposed that the BRS in CD3ε is responsible for anchoring the cytoplasmic tail of the resting TCR to the inner leaflet of the membrane lipid bilayer and that TCR activation releases this anchoring 13 . According to this model, the di-glycine motif could be important for information transfer to the motifs downstream of the BRS, including the PRS and the ITAM. Although we do not have experimental confirmation, our running hypothesis is that the G169A mutations have a structural impact on the cytoplasmic tail of CD3ε, rather than Gly169 being directly involved in the binding of an effector protein. Glycine residues are, after proline residues, the lowest amino acids on a scale of propensity to be found in α-helixes, whereas alanine residues are at the top 23 . Gly169 is preceded by an alanine, so it could be imagined that the G169A mutation allows the formation of half a turn of an α-helix, making the otherwise unstructured region adopt a structure less prone to transmitting conformational information from the N-terminus to the C-terminus in the cytoplasmic tail. One of the most striking consequences of the G169A mutation was the differential effect on the phosphorylation of specific tyrosine residues within the ITAMs of CD3ε and CD3ζ. The deficient phosphorylation of Tyr166 (N-terminal tyrosine) in the ITAM of CD3ε may be a consequence of impaired transduction of the conformational change, resulting in reduced exposure of the ITAM to phosphorylation by Lck. Interestingly, the effect on CD3ζ phosphorylation depended on the specific tyrosine residues, i.e. Tyr111 (ITAM ζb) and Tyr142 (ITAM ζc) did not seem to be affected. However, phosphorylation of Tyr83 (ITAM ζa) was inhibited in the presence of the G169A mutation. In our Western blots under non-reducing conditions, phospho-CD3ζ appeared as two distinct bands of low and high molecular weight, which could represent the dimers of the p21 and p23 forms of CD3ζ that have been studied for years 16 . Of note, a hierarchy of ITAM tyrosine phosphorylation has been proposed in which tyrosine residues in ITAMs ζb and ζc are first phosphorylated to form the p21 form, with ITAM ζa being the last to be phosphorylated to form the fully phosphorylated p23 form 16 . In the presence of the G169A mutation, we detected an impaired conversion of the low-phosphorylated to the high-phosphorylated form of CD3ζ, and this coincides with a poor phosphorylation of Tyr83, which we detected only in the fully phosphorylated form. These data suggest that the G169A mutation prevents the phosphorylation of ITAM ζa and, consequently, the full phosphorylation of CD3ζ. How does the G169A mutation selectively affect the phosphorylation of ITAM ζa? One clue could be the RK motif located downstream of the first tyrosine (Tyr166) of CD3ε that has been implicated in TCR-triggered recruitment of Lck 15 . In that paper, an effect of a double mutation (RKAA) on CD3ζ phosphorylation was described, although no differential effect on specific tyrosine residues was reported. On the other hand, it has also been shown that the BRS in CD3ε is involved in the recruitment of Lck 14 . The di-glycine motif described in this work is placed between the BRS and the RK motifs, therefore, a working model (Fig. 4 e) could be that G169, and the di-glycine motif, is necessary to interconnect the two sequence features in CD3ε involved in Lck recruitment, perhaps by allowing the cytoplasmic tail of CD3ε to have the sufficient flexibility to interconnect both features. Our data suggest that the di-glycine motif upstream of the PRS is required to recruit Lck, being this recruitment necessary for the phosphorylation of the ITAM ζa and full CD3ζ phosphorylation. Another consequence of the G169A mutation is the impairment of the phosphorylation of Lck at Ser59 by ERK in a feedback loop. However, the consequences of such impairment of Ser59 phosphorylation are not yet fully understood due to controversy surrounding its positive or negative role. For instance, initial observations suggest that Ser59 phosphorylation reduces Lck activity 19 and inhibits TCR signaling in Jurkat T cells expressing a phosphomimetic S59E Lck mutant 24 . Conversely, another study has shown that phosphorylation of Ser59 interferes with the interaction between Lck and the phosphatase SHP-1, preventing dephosphorylation of Tyr394 and subsequent Lck inactivation in thymocytes 25 . Moreover, blockade of the Erk1/2-Lck feedback loop, and hence Ser59 phosphorylation, rapidly terminates proximal TCR signaling 20 . In any case, the effect of the G169A mutation on Ser59 phosphorylation appears to be secondary to the effect on ITAM ζa phosphorylation, as the latter occurs with faster kinetics (a peak at 1 min versus a peak at 5 min of stimulation). Regardless, what is ultimately responsible for the defective T cell activation observed in late events (IL-2 release, CD69 and CD25 expression), our findings on the effects of the G169A mutation in CD3ε suggest that direct TCR signaling is a finely-tuned, stepwise process. This process involves an outside-in and N-terminal-to-C-terminal transfer of information within CD3ε, leading to selective impacts on specific residues not only of CD3ε but also of CD3ζ, and influencing the activity and phosphorylation of the associated tyrosine kinase Lck. These data support conformational models of intra-TCR signaling and challenge models based on simple molecular exclusion. Further research is needed to understand the structural significance of the glycine-to-alanine mutation at position 169. METHODS Cells. The human T-cell Jurkat line was maintained in complete RPMI 1640 supplemented with 5% fetal bovine serum (FBS, Sigma). Plasmids. The pGEX-4T1 derivative GST-SH3.1 containing the amino-terminal SH3 domain of Nck1 was kindly provided by R. Geha (Children’s Hospital, Harvard Medical School, Boston, MA). Point mutations in human CD3ε were made using the QuickChange-XL (Stragene). The FLAG-epitope was added to the C-terminus and the cDNA encoding human CD3ε was cloned into pSRα The GFP-expressing HIV vector pHRSIN-WPRE 26 was provided by Dr. J.A. Pintor (CABIMER-CSIC, Sevilla) wherein murine CD3ε cDNA replaced GFP to construct pHRSIN-mCD3ε. Generation of CD3ε knockout Jurkat cells by CRISPR/Cas9 To inactivate the endogenous Cd3e gene in Jurkat cells, we utilized the CRISPR/Cas9 system as described by Ran et al 27 . Briefly, sgRNAs specific to the target gene were designed using an online CRISPR Design tool ( http://tools.genome-engineering.org ). One set of sgRNAs was used targeting EXON 2 of the Cd3e gene (Suppl. Table 1). DNA oligos encoding the top and bottom sgRNA strands were hybridized and ligated into the pSpCas9(BB)-2A-GFP (PX458) vector. These vectors were then electroporated in circular form into Jurkat cells. Forty-eight hours post-electroporation, single cell sorting was performed to isolate individual CD3εKO Jurkat clones that were GFP + and CD3 − by flow cytometry. Generation of mutant CD3ε G169A Jurkat cells. First, vectors containing CD3ε mutations were generated using direct mutation PCR with primers listed in Supplementary Table 1. Once the mutations were obtained, the PSRalpha vector containing mutated CD3ε was electroporated into Jurkat CD3εKO cells. CD3ε mutant clones expressing TCR levels comparable to WT Jurkat cells were selected (Suppl. Fig. S1 ). Antibodies and other reagents. The rabbit anti-CD3ζ antiserum 488 has been described previously 28 . The other antibodies used were purchased as follows: hybridomas producing the anti-human CD3 mAb OKT3 from ATCC; the anti-phospho-CD3ζ (pTyr83), Anti-phospho-CD3ζ (pTyr142), anti-phospho-CD3ζ (pTyr111), anti-HA (12CA5) from Sigma; HRP Mouse Anti-Phosphotyrosine PY20 from BD biosciences; rabbit anti-mouse pERK (T202/Y204), anti-phospho Zap70 (Y319)/Syk(Y352), anti- phospho-Lck (Tyr505), anti-phospho-Lck (Y394), anti-phospho LAT (Y132), anti-phospho Src family (Y416) from Cell Signalling; anti-GST peroxidase conjugated from Rockland Immunochemicals; anti-phospho Lck (Y394) from RB Systems; anti-phospho Lck (Ser59), anti-CD3γ and anti-TCRα from Thermo Fisher scientific. T cell stimulation and flow cytometry. For CD69 and CD25 expression, 2x10 5 cells were analyzed 24 h after stimulation with different concentrations of anti-CD3 OKT3 or PMA and ionomycin. Anti-CD28 (0.1 µg/ml) was added for all anti-CD3 concentrations. After stimulation, cells were washed in PBS + 2%FBS and stained with anti-human CD69-FITC and CD25-APC (Invitrogen) antibodies for 30 min at 0ºC. Cells were then washed twice and analyzed by flow cytometry (FACS Canto II Becton Dickinson) and FlowJo software. Dead cells were excluded in all analyses by using DAPI. T cell stimulation and Western blotting Jurkat cells were resuspended in RPMI plus 20mM Hepes and left under starving conditions overnight. The MEK inhibitor U0126 (20 µM) was added when indicated. Cells were stimulated at different time points with anti-CD3 OKT3 antibody. After stimulation, cells were lysed in Brij96 lysis buffer containing protease and phosphatase inhibitors (0.33% Brij96, 140mM NaCl, 20mM tris-HCl (pH7.8), 10mM iodoacetamide, 1mM PMSF (phenylmethylsulfonyl fuoride), leupeptin (1µg/ml), aprotinin (1µg/ml), 1mM sodium orthovanadate, and 20mM sodium fuoride). For western blotting, the samples were resolved by SDS–polyacrylamide gel electrophoresis (PAGE). The recovered proteins were then transferred to a nitrocelullose membrane that was probed with different antibodies. Finally, the membrane was incubated with streptavidin-conjugated horseradish peroxidase (Southern Bio-technology) and developed by ECL (Pierce). Immunoprecipitation For imunoprecipitation, whole-cell lysates of the stimulated cells were incubated with anti-CD3 (OKT3) antibodies bound to protein G-Sepharose beads. The immunoprecipitated material was washed 5 times in Brij96 lysis buffer and finally resuspended in 20 µl of Laemmli sample buffer. Samples were resolved by SDS-PAGE and immunoblotting was carried out for the identification of total membrane proteins associated with OKT3. Pull-down assay. For the GST-Nck pull-down assay, cell lysates were incubated with purified recombinant protein consisting of glutathione-S-transferase (GST) fused to the SH3.1 domain of human Nck1 or glutathione-S-transferase (GST) at 4ºC for 2 hours. After washing 5 times, the pull down beads were boiled in Laemmli sample buffer and subjected to SDS-PAGE and then transferred by Western blot to a nitrocellulose membrane (Bio-Rad) which was probed with different antibodies. Detection of human IL-2 by ELISA IL-2 detection was performed using DuoSet® ELISA development system kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. The experiments were carried out in triplicates and in a 96-well ELISA plate and washes were carried out using Wash buffer (PBS with 0,05% Tween®-20) three times per wash. The plate was coated with α-hIL-2 capture antibody (4 µg/ml), sealed and incubated overnight. The coated antibody was discarded and the plate was washed. The plate was then incubated with Block buffer (PBS with 1% bovine serum albumin (BSA), 0,22 µm filtered) for 1 hour at room temperature. After washing, the 100 µl/well of the samples were plated. Dilutions of R-hIL-2 standards (as recommended by the manufacturer) were prepared in Reagent diluent (Tris-buffered saline (TBS) with 0,1% BSA, 0,05% Tween®-20, pH 7,2–7,4; 0,22 µm filtered) and made in duplicates for generation of standard curve. Plates were sealed and incubated 2 hours at room temperature. The plate was washed and the detection antibodies g-α-hIL-2-biotin (BIO) (100 ng/ml) diluted in Reagent diluent were incubated for 2 hours at room temperature. The plate was washed and incubated with streptavidin-horseradish peroxidase (STV-HRP) for 20 min in the dark at room temperature. Substrate solution (1:1 mix of 3,3',5,5'-tetramethylbenzidine (TMB) and H 2 O 2 ) was added, after washing, and incubated up to 20 min in the dark at room temperature. 50 µl/well Stop solution (2N H 2 SO 4 ) was added to stop the reaction and optical density was measured using iMark™ microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA) at 450nm with subtractive correction at 595nm. Declarations Competing interests The authors declare that they have no competing interests. Author Contribution RT carried out most of the experiments; ERB and NMB generated mutations, prepared constructs and selected clones; AL carried out some of the immunoblottings; BA conceived the project, directed the research and wrote the manuscript, which was edited and revised by RT, ERB and AL. Acknowledgements We are indebted to Valentina Blanco and Tania Gómez for their expert technical assistance. This work was supported by Grant PID2022-136745OB-I00 funded by AEI/ 10.13039/501100011033 and, by the “European Union NextGenerationEU/PRTR”, and by Grant P2022/BMD7209 (INTEGRAMUNE-CM) from the ‘Comunidad de Madrid’. Data Availability The data supporting the conclusions of this article are presented within article and its additional files. References Beddoe, T., Chen, Z., Clements, C.S., Ely, L.K., Bushell, S.R., Vivian, J.P., Kjer-Nielsen, L., Pang, S.S., Dunstone, M.A., Liu, Y.C., et al. (2009). Antigen ligation triggers a conformational change within the constant domain of the alphabeta T cell receptor. Immunity. 30 , 777 – 88. Epub 2009 May 21. Hawse, W.F., Champion, M.M., Joyce, M.V., Hellman, L.M., Hossain, M., Ryan, V., Pierce, B.G., Weng, Z., and Baker, B.M. (2012). Cutting Edge: Evidence for a Dynamically Driven T Cell Signaling Mechanism. The Journal of Immunology 188 , 5819–5823. https://doi.org/10.4049/jimmunol.1200952 . Natarajan, K., McShan, A.C., Jiang, J., Kumirov, V.K., Wang, R., Zhao, H., Schuck, P., Tilahun, M.E., Boyd, L.F., Ying, J., et al. (2017). An allosteric site in the T-cell receptor Cbeta domain plays a critical signalling role. Nat Commun. 8:15260. , 10.1038/ncomms15260 . Rangarajan, S., He, Y., Chen, Y., Kerzic, M.C., Ma, B., Gowthaman, R., Pierce, B.G., Nussinov, R., Mariuzza, R.A., and Orban, J. (2018). Peptide–MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites. Journal of Biological Chemistry 293 , 15991–16005. https://doi.org/10.1074/jbc.RA118.003832 . Gil, D., Schamel, W.W., Montoya, M., Sanchez-Madrid, F., and Alarcon, B. (2002). Recruitment of Nck by CD3 epsilon reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell 109 , 901 – 12. Gil, D., Schrum, A.G., Alarcon, B., and Palmer, E. (2005). T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes. J Exp Med 201 , 517–22. Tunnacliffe, A., Buluwela, L., and Rabbitts, T.H. (1987). Physical linkage of three CD3 genes on human chromosome 11. Embo J. 6 , 2953–7. Dzialo, R.C., and Cooper, M.D. (1997). An amphibian CD3 homologue of the mammalian CD3 gamma and delta genes. Eur J Immunol. 27 , 1640–7. Love, P.E., and Hayes, S.M. (2010). ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb Perspect Biol. 2 , a002485. Epub 2010 Apr 28. Bu, J.Y., Shaw, A.S., and Chan, A.C. (1995). Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance. Proc Natl Acad Sci U S A. 92 , 5106–10. Isakov, N., Wange, R.L., Burgess, W.H., Watts, J.D., Aebersold, R., and Samelson, L.E. (1995). ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity. J Exp Med. 181 , 375–80. Borroto, A., Abia, D., and Alarcon, B. (2014). Crammed signaling motifs in the T-cell receptor. Immunol Lett. 161 , 113–7. doi: 10.1016/j.imlet.2014.05.007 . Epub 2014 May 27. Xu, C., Gagnon, E., Call, M.E., Schnell, J.R., Schwieters, C.D., Carman, C.V., Chou, J.J., and Wucherpfennig, K.W. (2008). Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3varepsilon Cytoplasmic Tyrosine-Based Motif. Cell. 135 , 702–13. Li, L., Guo, X., Shi, X., Li, C., Wu, W., Yan, C., Wang, H., Li, H., and Xu, C. (2017). Ionic CD3 – Lck interaction regulates the initiation of T-cell receptor signaling. Proc. Natl. Acad. Sci. U.S.A. 114 . https://doi.org/10.1073/pnas.1701990114 . Hartl, F.A., Beck-Garcìa, E., Woessner, N.M., Flachsmann, L.J., Cárdenas, R.M.-H.V., Brandl, S.M., Taromi, S., Fiala, G.J., Morath, A., Mishra, P., et al. (2020). Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function. Nat Immunol 21 , 902–913. https://doi.org/10.1038/s41590-020-0732-3 . Van Oers, N.S.C., Tohlen, B., Malissen, B., Moomaw, C.R., Afendis, S., and Slaughter, C.A. (2000). The 21- and 23-kD forms of TCRζ are generated by specific ITAM phosphorylations. Nat Immunol 1 , 322–328. https://doi.org/10.1038/79774 . Bommhardt, Schraven, and Simeoni (2019). Beyond TCR Signaling: Emerging Functions of Lck in Cancer and Immunotherapy. IJMS 20 , 3500. https://doi.org/10.3390/ijms20143500 . Di Bartolo, V., Mège, D., Germain, V., Pelosi, M., Dufour, E., Michel, F., Magistrelli, G., Isacchi, A., and Acuto, O. (1999). Tyrosine 319, a Newly Identified Phosphorylation Site of ZAP-70, Plays a Critical Role in T Cell Antigen Receptor Signaling. Journal of Biological Chemistry 274 , 6285–6294. https://doi.org/10.1074/jbc.274.10.6285 . Watts, J.D., Sanghera, J.S., Pelech, S.L., and Aebersold, R. (1993). Phosphorylation of serine 59 of p56lck in activated T cells. Journal of Biological Chemistry 268 , 23275–23282. https://doi.org/10.1016/S0021-9258(19)49459-3 . Poltorak, M., Arndt, B., Kowtharapu, B.S., Reddycherla, A.V., Witte, V., Lindquist, J.A., Schraven, B., and Simeoni, L. (2013). TCR activation kinetics and feedback regulation in primary human T cells. Cell Commun Signal 11 , 4. https://doi.org/10.1186/1478-811X-11-4 . Nika, K., Soldani, C., Salek, M., Paster, W., Gray, A., Etzensperger, R., Fugger, L., Polzella, P., Cerundolo, V., Dushek, O., et al. (2010). Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity. 32 , 766 – 77. Epub 2010 Jun 11. Bettini, M.L., Chou, P.-C., Guy, C.S., Lee, T., Vignali, K.M., and Vignali, D.A.A. (2017). Cutting Edge: CD3 ITAM Diversity Is Required for Optimal TCR Signaling and Thymocyte Development. The Journal of Immunology 199 , 1555–1560. https://doi.org/10.4049/jimmunol.1700069 . Pace, C.N., and Scholtz, J.M. (1998). A helix propensity scale based on experimental studies of peptides and proteins. Biophys J. 75 , 422–7. Dutta, D., Barr, V.A., Akpan, I., Mittelstadt, P.R., Singha, L.I., Samelson, L.E., and Ashwell, J.D. (2017). Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol 18 , 196–204. https://doi.org/10.1038/ni.3640 . Štefanová, I., Hemmer, B., Vergelli, M., Martin, R., Biddison, W.E., and Germain, R.N. (2003). TCR ligand discrimination is enforced by competing ERK positive and SHP-1 negative feedback pathways. Nat Immunol 4 , 248–254. https://doi.org/10.1038/ni895 . Demaison, C., Parsley, K., Brouns, G., Scherr, M., Battmer, K., Kinnon, C., Grez, M., and Thrasher, A.J. (2002). High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther. 13 , 803–13. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8 , 2281–2308. https://doi.org/10.1038/nprot.2013.143 . San Jose, E., Sahuquillo, A.G., Bragado, R., and Alarcon, B. (1998). Assembly of the TCR/CD3 complex: CD3 epsilon/delta and CD3 epsilon/gamma dimers associate indistinctly with both TCR alpha and TCR beta chains. Evidence for a double TCR heterodimer model. Eur J Immunol 28 , 12–21. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4594242","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":328304731,"identity":"55566994-4cdc-4d15-931e-2bf51d308bf2","order_by":0,"name":"Rut Tercero","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":false,"prefix":"","firstName":"Rut","middleName":"","lastName":"Tercero","suffix":""},{"id":328304734,"identity":"4bb6f923-2897-49d9-beee-78761d164c6b","order_by":1,"name":"Elena R. Bovolenta","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"R.","lastName":"Bovolenta","suffix":""},{"id":328304736,"identity":"548a7dd0-e716-427b-8bba-84aa3e2218b2","order_by":2,"name":"Nadia Martín-Blanco","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Martín-Blanco","suffix":""},{"id":328304737,"identity":"67ab8e81-eaf8-4e14-ad48-346a0fd4bdfc","order_by":3,"name":"Alice Lombard","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":false,"prefix":"","firstName":"Alice","middleName":"","lastName":"Lombard","suffix":""},{"id":328304738,"identity":"a373443e-dff3-40de-8b19-f10a372ff8c0","order_by":4,"name":"Balbino Alarcón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYBACAwbmBjCDn4EHTPMQoYURokWygWQtBgcIK4YAc/aDjZ8ramzyjI+fPbqBoaZOhr+B+fAHfFosexKbJc8cSys2O5OXdoPh2GEeiQNsaRJ4HXYgsUGyseFw4rYbPGY3GBsO8Bgw8Jjh98v5h80/QVo2zwBrqQNq4f+M12EGNxLbwLZskABrYQbZwoDXYZYzHrZZNhxLS5wB8ksCyC+H2czwajHnTz58s6HGJrG//eyxGx9q6uz525sf43UYKkgAEczEqx8Fo2AUjIJRgAMAAFduSmOYadjeAAAAAElFTkSuQmCC","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa","correspondingAuthor":true,"prefix":"","firstName":"Balbino","middleName":"","lastName":"Alarcón","suffix":""}],"badges":[],"createdAt":"2024-06-17 12:51:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4594242/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4594242/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60603296,"identity":"cd47ec8b-10ba-447e-8798-eb8de093b75e","added_by":"auto","created_at":"2024-07-18 16:22:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":346632,"visible":true,"origin":"","legend":"\u003cp\u003eMutation to alanine of the di-glycine motif in the cytoplasmic tail of CD3ε impairs TCR-triggered T cell activation. \u003cstrong\u003ea,\u003c/strong\u003e Alignment of the amino acid sequences found in the cytoplasmic domain of CD3ε of vertebrates. Sequences were retrieved from the NCBI database using the elephant shark CD3ε amino acid sequence (gi: 398692755) as the query. Sequence redundancy was reduced by removing sequences that shared more than a 70% identity. The remaining sequences were aligned as described\u003csup\u003e12\u003c/sup\u003e and displayed using the Jalview program with the default color scheme for ClustalW and auto-calculated consensus logo display. The font size of the amino acid symbol in the consensus logo indicates the degree of conservation in all species. Described sequence motifs are annotated at the bottom. These include: the Immunoreceptor Tyrosine-based Activation Motif (ITAM), the Proline-Rich Sequence (PRS), the Basic amino acid-rich sequence (BRS), the Arginine-Lysine RK motif, the Phosphotyrosine-Binding domain (PTB), the Endoplasmic Reticulum Retention sequence (ERR), and the poly-Glycine motif. \u003cstrong\u003eb,\u003c/strong\u003e Cartoon display of the structure of the cytoplasmic tail of murine CD3ε determined by NMR\u003csup\u003e13\u003c/sup\u003e according to PDB=2K4F. The PRS is highlighted in magenta and the ITAM in green colors. Some highly conserved amino acids, according to panel \u003cu\u003ea\u003c/u\u003e, are indicated with arrows. \u003cstrong\u003ec,\u003c/strong\u003e IL-2 release into the culture medium upon stimulation of human Jurkat T cells, bearing the indicated mutations in CD3ε, with different concentrations of the anti-CD3 antibody OKT3. A Jurkat CD3ε knockout (εKO) cell line was reconstituted with either wild-type CD3 (WT) or CD3ε bearing the indicated point mutations in the cytoplasmic tail. Data are represented as the mean ± SEM of biological triplicates. Statistical significance was assessed using a Two-way ANOVA Dunnett’s multiple comparisons test. \u003cstrong\u003ed,\u003c/strong\u003e CD69 expression induced upon stimulation of WT Jurkat T cells, or Jurkat cells bearing either the G169A or G170A mutations, with the indicated concentrations of OKT3. Stimulation with a TCR-independent stimulus, PMA+ionomycin (P+I), was carried out in parallel. Data are represented as the mean ± SEM of the percentage of CD69+ cells in triplicate. Statistical significance was assessed using a Two-way ANOVA Dunnett’s multiple comparisons test. \u003cstrong\u003ee,\u003c/strong\u003e CD25 expression induced upon stimulation of WT Jurkat T cells, or Jurkat cells bearing either the G169A or G170A mutations, with the indicated concentrations of OKT3. Data are represented as the mean ± SEM of the mean fluorescence intensity (MFI) in triplicate. Statistical significance was assessed using a Two-way ANOVA Dunnett’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"Figure1421.png","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/521d8fa0eba725218c542bb7.png"},{"id":60603989,"identity":"214a219a-e461-473d-9de5-1212a2a50e0c","added_by":"auto","created_at":"2024-07-18 16:30:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1091133,"visible":true,"origin":"","legend":"\u003cp\u003eThe G169A mutation impairs the TCR-triggered tyrosine phosphorylation at specific sites of CD3ε and CD3ζ. \u003cstrong\u003ea,\u003c/strong\u003eImmunoprecipitation with the anti-CD3 antibody OKT3 and Western blotting of the TCR in WT and G169A Jurkat T cells after stimulation with 10 µg/ml OKT3 for the indicated time points. Blotted membranes were probed with an antibody specific for the first tyrosine (Tyr166) of the ITAM of CD3ε and with an antibody specific for total CD3ζ. The positions of molecular weight markers run in parallel are indicated to the left. Bar plot to the right show the mean ± SEM of densitometric scans of three independent triplicates run at different times. Statistical significance was assessed using a paired t-test comparing all time points in parallel. \u003cstrong\u003eb,\u003c/strong\u003eCartoon illustrating the positions of the 6 tyrosine residues of the 3x2 ITAMs of the CD3ζdimer. \u003cstrong\u003ec,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points. The blotted membrane was probed with peroxidase-coupled anti-phosphotyrosine antibody PY20. The possible identities of the specifically induced tyrosine-phosphorylated proteins are indicated by arrows to the right. \u003cstrong\u003ed,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points and immunoblotted with an anti-phospho-CD3ζ(Tyr142)-specific antibody. Line plot below represents the result of densitometric scans quantified as in panel \u003cu\u003ea\u003c/u\u003e. \u003cstrong\u003ee-g,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points. Blotted membranes were probed with site-specific anti-phospho-CD3ζ antibodies (e-f) or with an anti-total anti-CD3ζ(g).\u003c/p\u003e","description":"","filename":"Figure1422.png","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/7b1593baff9af29ed9896278.png"},{"id":60603301,"identity":"669890de-aafc-4893-8b3e-b87add164c93","added_by":"auto","created_at":"2024-07-18 16:22:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":718185,"visible":true,"origin":"","legend":"\u003cp\u003eThe G169A mutation impairs TCR-triggered phosphorylation of proximal TCR effectors, including Ser59 of Lck by ERK. \u003cstrong\u003ea,\u003c/strong\u003e Cartoon illustrating some of the players and phosphorylation sites involved in TCR-proximal signaling. The active TCR is phosphorylated in several tyrosine residues within its ITAMs by the tyrosine kinase Lck. Lck possesses an activatory autophosphorylation site at Tyr394 and an inhibitory site at Tyr505, which is phosphorylated by Csk. The tyrosine kinase ZAP70 is recruited to the active TCR by binding to the previously phosphorylated ITAMs. TCR-bound ZAP70 is phosphorylated by Lck at Tyr319. Binding of ZAP70 to the TCR and phosphorylation by Lck activate ZAP70, which subsequently phosphorylates downstream effectors including LAT, an adaptor protein that recruits many others and is responsible for the activation of phospholipase C, also involved in the activation of the Raf-ERK MAP kinase pathway. Activated ERK phosphorylates Lck at residue Ser59, establishing a feedback loop. \u003cstrong\u003eb\u0026amp;c,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points. The blotted membrane was probed with specific antibodies for the proteins and phosphorylation sites indicated. Immunoblotting with an anti-CD3ζantibody was used as a loading control. The identities of the specifically detected proteins are indicated by arrows to the right. \u003cstrong\u003ed,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points. The blotted membrane was probed with an antibody for the activating phosphorylation site Tyr416 in Src, which is equivalent to the activating site Tyr394 in Lck. Immunoblotting with an anti-CD3ζ antibody was used as a loading control. Arrows on the right point out two distinct protein bands corresponding to two forms of Lck of apparent high and low molecular weight. \u003cstrong\u003ee,\u003c/strong\u003eInhibition of Lck Ser59 phosphorylation by the inhibitor UO126 of the ERK upstream activator MEK. Western blot of postnuclear whole-cell lysates of WT Jurkat cells pre-incubated with 20 µM UO126 or just the vehicle (DMSO) and stimulated with 10 µg/ml OKT3 for the indicated time points. The arrow on the right points out the position of Lck with high apparent molecular weight found in panel \u003cu\u003ec\u003c/u\u003e. Immunoblotting with an anti-CD3ζ antibody was used as a loading control. \u003cstrong\u003ef,\u003c/strong\u003e Western blot of postnuclear whole-cell lysates of WT and G169A Jurkat cells stimulated with 10 µg/ml OKT3 for the indicated time points. The blotted membrane was probed with an antibody for Lck phosphorylated on Ser59. The membrane was subsequently reprobed with anti-Lcl(Tyr394) to indicate the positions of the high and low molecular weight forms of Lck. Immunoblotting with an anti-CD3ζ antibody was used as a loading control.\u003c/p\u003e","description":"","filename":"Figure1423.png","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/9122525c7fe4e386e4492244.png"},{"id":60603300,"identity":"f39a6a9b-c176-4d70-9e5f-a66246a9ec38","added_by":"auto","created_at":"2024-07-18 16:22:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":602888,"visible":true,"origin":"","legend":"\u003cp\u003eThe G169A mutation impairs the transmission of outside-in conformational changes in the TCR, resulting in the exposure of the PRS in CD3ε for binding to the SH3.1 domain of Nck. \u003cstrong\u003ea,\u003c/strong\u003e Cartoon illustrating how TCR triggering with a stimulatory anti-CD3 antibody causes the TCR to adopt an Active conformation, manifested by the exposure of a hidden polyproline sequence (PRS) in the tail of CD3ε (blue square in the Resting TCR and yellow triangle in the Active TCR). Such exposure of the PRS makes the TCR competent to bind a GST-SH3.1 fusion protein in pull-down assays. \u003cstrong\u003eb,\u003c/strong\u003e Western blot after pull-down (Pd) with GST-SH3.1 beads from lysates of WT and G169A Jurkat cells stimulated for the indicated time points with anti-CD3 antibody. The captured proteins were resolved by SDS-PAGE under non-reducing conditions (i.e., in the absence of disulfide bridge reducing reagents), transferred to a membrane, and blots were probed sequentially with an anti-CD3ζ antibody, an anti-CD3γ antibody, and anti-TCRα antibody. The positions of molecular weight markers run in parallel are indicated on the left. The CD3ζ homodimer runs in these conditions as a 32 kD band, while the TCRα/β heterodimer runs as a 90 kD band. A western blot with anti-GST antibody was carried out as a loading control of the pull-down assay. The position of the GST-SH3.1 fusion protein is indicated. \u003cstrong\u003ec,\u003c/strong\u003e Western blot after pull-down (Pd) with GST-SH3.1 beads of cell lysates from WT and G169A Jurkat cells stimulated for the indicated time points with anti-CD3 antibody. Blotting membranes were incubated with the anti-phospho-CD3ζ(Tyr142) and with the anti-phospho-Lck(Tyr394) and Lck(Ser59) antibodies. Western blots on whole cell lysates with anti-phospho-Lck(Tyr394) and anti-CD3ζ antibodies were used as loading controls. \u003cstrong\u003ed,\u003c/strong\u003eWorking model of how stimulation of the TCR with anti-CD3 binding to the extracellular domains transmits the Active conformation to the cytoplasmic tail of CD3ε, resulting in the induced recruitment of Nck to the TCR. Transmission of the Active conformation to the PRS for Nck binding is impaired if Glycine 169 is mutated to alanine. Glycine 169 is required for the recruitment of Lck to the cytoplasmic tail of CD3ε, for phosphorylation of the ITAM of CD3ε and for phosphorylation of the membrane-proximal ITAM (ITAM ζa) of CD3ζ by Lck.\u003c/p\u003e","description":"","filename":"Figure1424.png","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/7c32015f643f83df2fd8c792.png"},{"id":60868813,"identity":"329d2a79-a3db-4eed-abfe-4a3a9797d62b","added_by":"auto","created_at":"2024-07-23 04:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3475825,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/0dfc28f3-6b95-412d-95f3-ac0b80be0e5b.pdf"},{"id":60603298,"identity":"a783c007-597e-4247-99e1-715970b4d88f","added_by":"auto","created_at":"2024-07-18 16:22:34","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9557,"visible":true,"origin":"","legend":"","description":"","filename":"SupplTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/01942383fd647c04040ab0e8.xlsx"},{"id":60603302,"identity":"17966128-c0d8-4fac-a2a6-1ecc5ef2010a","added_by":"auto","created_at":"2024-07-18 16:22:34","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12749133,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4594242/v1/64e02a85988f1dea12aabdbd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A di-glycine motif in the cytoplasmic tail of CD3ε required for transmission of allosteric changes in the TCR and full CD3ζ phosphorylation","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe T cell antigen receptor (TCR) is responsible for recognizing antigens and can be considered the foundation of the adaptive immune system due to the ability of T cells to orchestrate the type and intensity of the immune response to pathogen-encoded antigens. In mammals, the majority of T cells (αβ T cells) recognize antigens in the form of peptides inserted into a groove formed by molecules of the major histocompatibility complex (MHC). αβ T cells bearing a TCR capable of recognizing the complex of the antigenic peptide and an MHC allele (pMHC complex) with sufficient affinity are activated and enter a program of proliferation and differentiation. This process generates various effector and memory T cells that are responsible for mounting an immune response to eliminate the antigen and retaining antigen memory for faster and stronger responses to future encounters with the same antigen.\u003c/p\u003e \u003cp\u003eThe TCR is a multi-subunit complex consisting of the TCRα and TCRβ subunits, which are responsible for recognizing pMHC through their variable sequence regions, and the invariable CD3γ, CD3δ, CD3ε, and CD3ζ (also known as CD247) subunits. The TCRα and TCRβ subunits have very short cytoplasmic tails and are, in principle, incapable of interacting with cytoplasmic effector proteins. In contrast, CD3 subunits have longer cytoplasmic tails that have been shown to recruit effector proteins such as the tyrosine kinase Lck, the tyrosine kinase ZAP70, and the adapter protein Nck upon pMHC binding to the TCRα/β subunits. Somehow, the information about pMHC binding must flow from the TCRα/β subunits to the cytoplasmic tails of the CD3 subunits. The most obvious mechanism would be that of allosteric changes resulting from conformational changes that transfer outside-in information about pMHC binding from the TCRα/β ectodomains to the CD3 ectodomains and transmembrane regions and from these to the cytoplasmic tails. Some structural data have been generated supporting the existence of ligand-induced conformational changes in the ectodomains of the TCR\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eHowever, the strongest evidence to date for conformational changes in the TCR has come from the observation that TCR triggering by pMHC or agonistic anti-CD3 antibodies induces the exposure of various motifs in the cytoplasmic tails. The earliest evidence was the finding that a proline-rich sequence (PRS) unique to the tail of CD3ε does not adopt a conformation prone to bind the N-terminal SH3 domain (SH3.1) of Nck when the TCR is at rest\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Ligation of the TCRα/β or CD3 ectodomains with agonistic antibodies or binding of cognate pMHC results in the PRS adopting a conformation able to bind the SH3.1 domain of Nck\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These results suggest that there must be an outside-in mechanism for transmitting allosteric changes to the PRS of CD3ε.\u003c/p\u003e \u003cp\u003eThe CD3γ, CD3δ, and CD3ε subunits are located within a 30 Mb region on human chromosome 11 (and on chromosome 9 in mice), with CD3γ and CD3δ positioned head-to-head and separated by 1.6 kb\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. These CD3γ and CD3δ subunits result from a relatively recent duplication, as only mammals have two distinct genes for them, while birds and lower vertebrates (reptiles, amphibians, and fish) possess only one gene encoding a glycoprotein with intermediate homology to CD3γ and CD3δ8. CD3ε is already present in cartilaginous fish and has remained highly conserved. This conservation likely reflects its role in forming dimers with both CD3γ and CD3δ in mammals, which are at least partially redundant.\u003c/p\u003e \u003cp\u003eThe cytoplasmic tails of the CD3 subunits contain either one (CD3γ, CD3δ, and CD3ε) or three (CD3ζ) tyrosine- and leucine-based motifs with the consensus sequence YxxL/I (x)6\u0026ndash;9 YxxL/I. These motifs, known as ITAMs (Immunoreceptor Tyrosine-based Activation Motifs), are rapidly phosphorylated upon TCR activation by the src family tyrosine kinases Lck and Fyn\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. When ITAMs are phosphorylated at both tyrosines, they become high-affinity (nM) docking sites for proteins with two SH2 domains in tandem, such as the Syk family tyrosine kinases, Syk and ZAP70\u003csup\u003e10,11\u003c/sup\u003e. The presence of multiple ITAMs in a single TCR complex (up to 10, considering one CD3ζ homodimer and two CD3ε subunits) has sparked debate over whether this multiplicity serves purely to amplify signals or also has qualitative effects. The hypothesis that each ITAM can bind different sets of signaling proteins is supported by the fact that the amino acids around the two YxxL/I motifs and those in the spacer between them differ for each ITAM but have been conserved throughout vertebrate evolution. Interestingly, a comparison of the amino acid sequences of the cytoplasmic tail of CD3ε in vertebrates shows that the most highly conserved feature is not the ITAM but the PRS\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Other highly conserved amino acids have also been identified.\u003c/p\u003e \u003cp\u003eIn this work, we made point mutations in several evolutively conserved amino acids in the CD3ε cytoplasmic tail and identified Gly169 as an important amino acid for TCR signaling. Its mutation alters the earliest detectable activation events, including induced exposure of the PRS, phosphorylation of some ITAMs in CD3ζ, and recruitment of Lck to the TCR. We discuss the relevance of these findings to our understanding of how the TCR transduces activation signals.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eAn evolutionarily conserved glycine motif located between the Basic-Rich Sequence (BRS) and the Proline-Rich Sequence (PRS) in the cytoplasmic tail of CD3ε is essential for T cell activation induced by the T Cell Receptor (TCR).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA comparison of the amino acid sequences in the cytoplasmic tail of CD3ε reveals a remarkable pattern of conservation across ontogeny, spanning from cartilaginous fishes to mammals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The most conserved feature is the Proline-Rich Sequence (PRS, PxxPxPDY), followed by the tyrosine residues of the Immunoreceptor Tyrosine-based Activation Motif (ITAM). Other sequences previously reported to be crucial for outside-in signal transmission within the TCR are also preserved, including the Basic-Rich Sequence (BRS)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e and the RK motif involved in recruiting Lck\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, here are other conserved amino acid residues located between the BRS and the PRS. Notably, we noticed the presence of a conserved proline residue (Pro161 in human CD3ε) and one or more glycine residues, forming a di-glycine motif (Gly169-Gly170 in human CD3ε), present in all vertebrates and conserved in mammals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Characterization of the structure of the cytoplasmic tail of murine CD3ε by Nuclear Magnetic Resonance (NMR) (PDB\u0026thinsp;=\u0026thinsp;2K4F) revealed a random coil throughout the 57 amino acids of the polypeptide, except for two turns of an α-helix around the C-terminal tyrosine of the ITAM, an α-helix turn at the end of the PRS and N-terminal tyrosine of the ITAM, and an α-helix turn immediately after Pro161 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The residue corresponding to Gly169 is located within a long unstructured sequence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess whether mutation to alanine of different residues in human CD3ε affected the TCR's ability to activate T cells in response to ligation with an anti-CD3 antibody, we first generated a CRISPR/Cas9 full knockout of CD3ε in the Jurkat T cell line. Subsequently, the resulting CD3εKO cell line was transfected with either wild-type CD3ε or alanine mutants corresponding to residues Arg155 and Arg156 of the BRS, residue Pro161, residues Gly169 and Gly170 of the di-glycine motif, and residues Arg192 and Lys193 of the RK motif. Stable clones were selected, and those exhibiting TCR expression at the cell surface similar to that of wild-type Jurkat cells (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e in the Supplementary Information) were chosen for functional analysis. To this end, all Jurkat clones were stimulated with various concentrations of anti-CD3 antibody, and IL-2 present in the supernatant was measured by ELISA. As expected, the CD3εKO line failed to produce IL-2 in response to anti-CD3 stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). All mutants significantly reduced IL-2 production compared to the wild-type control. Of note, cells bearing the G169A mutation exhibited the least responsiveness to anti-CD3 stimulation, releasing IL-2 at levels as low as the CD3εKO control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In a separate set of experiments, Jurkat CD3εKO cells were transduced with lentiviral constructs expressing either wild-type CD3ε or the G169A or G170A mutants, and the resulting polyclonal population was stimulated with different doses of anti-CD3. The response to anti-CD3 stimulation was evaluated based on the expression of CD69 and CD25 as activation markers. Cells bearing either glycine mutation showed deficient responses to TCR triggering (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These results demonstrate that mutation of glycine residues 169 and 170 to alanine in CD3ε diminishes the human TCR's ability to respond to stimulation with an anti-CD3 antibody.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe G169A mutation in CD3ε has a differential impact on the phosphorylation of specific tyrosine residues in CD3ζ.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo begin dissecting the mechanisms by which mutation of the di-glycine motif impairs TCR-triggered T cell activation, we first investigated some of the earliest events after TCR triggering, namely the tyrosine phosphorylation of the CD3ε and CD3ζ ITAMs. We focused our analysis on the G169A mutation, given that this has a stronger impact on IL-2 production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In anti-CD3 immunoprecipitates, we observed that the phosphorylation of the N-terminal tyrosine (Tyr166) of CD3ε was significantly inhibited by the G169A mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). While CD3ε has just two phosphorytable tyrosine residues in its single ITAM, CD3ζ contains three ITAMs in its cytoplasmic tail, accounting for a total of six phosphorylatable tyrosines\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A diagram illustrating the residue numbers for these six tyrosine residues within the three ITAMs of human CD3ζ is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. A deficiency in CD3ζ phosphorylation in Jurkat cells expressing the G169A mutant was noticed in Western blots of postnuclear supernatants of whole-cell lysates with a pan-phosphotyrosine-specific antibody (Fig, 2c). In addition to a putative defect on the tyrosine phosphorylation of CD3ζ, a clear defect in the phosphorylation of other proteins was noticed. According to their mobility in SDS-PAGE, those proteins could correspond to phospho-ZAP70 and phospho-Lck (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Interestingly, the putative phospho-CD3ζ bands showed distinct high- and low-molecular-weight forms, and the G169A mutant reduced the phosphorylation of the upper form of apparent high molecular weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). To determine if the G169A mutation in CD3ε primarily affected the phosphorylation of CD3ζ to its highest molecular weight form, we examined the effect of the mutations on the tyrosine phosphorylation of CD3ζ using a phospho-specific antibody for Tyr142 (ITAM ζc). Tyrosine-phosphorylated CD3ζ in Jurkat cells with a wild-type TCR appeared as two protein bands of different molecular weight, with the slowest mobility one presumably corresponding to the fully phosphorylated form. While the G169A mutation did not abolish tyrosine phosphorylation of CD3ζ, it did lead to a noticeable reduction in the ratio of highly phosphorylated to low-phosphorylated forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further examined the effects of the G169A mutation on CD3ζ phosphorylation using two additional available antibodies specific for Tyr83 (ITAM ζa) and Tyr111 (ITAM ζb) in postnuclear total cell lysates. Western blot analysis with the Tyr111-specific antibody revealed a protein band corresponding to the low-molecular weight form and a faint band corresponding to the high-molecular weight form, although only in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Intriguingly, western blotting with the Tyr83-specific antibody showed only one protein band corresponding to the high-molecular-weight form in wild-type cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). This single protein band was reduced in Jurkat cells expressing the G169A mutation compared with WT. Altogether, these findings suggest that the low-molecular weight form of phospho-CD3ζ is phosphorylated at Tyr111 and Tyr142 but not at Tyr83, whereas the high-molecular-weight form is phosphorylated at Tyr83, Tyr142, and, to a lesser extent, at Tyr111. Mutation G169A inhibited the phosphorylation of CD3ζ at Tyr83 but not at Tyr111 or Tyr142 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed-f), indicating that the mutation in CD3ε specifically impairs the phosphorylation of Tyr83 in the first ITAM of CD3ζ. Western blotting with antibody specific for total CD3ζ, phosphorylated and unphosphorylated, served as a loading control and to indicate that the unphosphorylated CD3ζ has the mobility of low apparent molecular weight one (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The effect on the phosphorylation of the other three tyrosine residues, for which suitable antibodies were not available, remains unknown. Nevertheless, these results suggest that the G169A mutation has a specific impact on a tyrosine residue located in the upstream, membrane-proximal ITAM of CD3ζ.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe G169A mutation impairs proximal TCR signaling and the phosphorylation of Lck at Ser59 by ERK.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the consequences of the G169A mutation on TCR signaling downstream of the TCR, we decided to measure the phosphorylation of the tyrosine kinases Lck and Zap70, which are tightly associated with early signal transmission. A general cartoon depicting the hierarchy of phosphorylation events following TCR activation is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The activated TCR becomes phosphorylated on the tyrosine residues of its ITAMs by Lck. Different tyrosine phosphorylation sites have been identified in Lck, with two of the most important being the autophosphorylation site Tyr394 in its catalytic domain, which activates Lck, and the inhibitory site Tyr505 located at the negative regulatory tail\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Double-site tyrosine phosphorylation of the ITAMs by Lck creates docking sites for the cytoplasmic tyrosine kinase Zap70, which becomes recruited to the TCR. Once bound to the TCR, Zap70 becomes phosphorylated, including at the activating site Tyr319\u003csup\u003e18\u003c/sup\u003e. Active Zap70 phosphorylates, among other targets, the membrane-bound adapter protein LAT, which serves as a docking site for PLCγ1. Activation of PLCγ1 leads to activation of, among others, the Raf-ERK MAP kinase pathway. Raf activates the dual kinase MEK, which phosphorylates ERK at the Thr202 and Tyr204 sites. Active ERK is part of a feedback loop since it phosphorylates Ser59 in the unique region of Lck\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The effect of such phosphorylation on Ser59 is debated, with some data supporting a reduction in Lck activity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and others supporting enhanced activity by inhibiting the recruitment of the phosphatase SHP-1\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon stimulation with anti-CD3 antibody in Jurkat cells bearing the G169A mutation phosphorylation at Tyr319 of Zap70 was strongly inhibited compared to that found in wild-type CD3ε Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Similar results were obtained for phosphorylation of LAT at Tyr132 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec); phosphorylation of ERK by MEK was inhibited as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWe studied the effect of the G169A mutation on the phosphorylation of Lck at both the activating site Tyr394 and the inhibitory site Tyr505 in total cell lysates. We found that stimulation of both WT and G169A cells with anti-CD3 did not increase the total content of phospho-Lck, regardless of the anti-phosphosite antibody used (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), consistent with previous findings\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, there was a clear increase in the apparent molecular weight of Lck induced by anti-CD3 stimulation. Specifically, a higher molecular weight band appeared after 5 minutes of stimulation, rather than at the earlier time point of 1 minute, and this band increased in abundance after stimulation at the expense of a low-molecular weight band (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The G169A mutation reduced the stimulation-induced increase in the upper high-molecular weight form at the expense of the low-molecular weight form, as visualized by a diminished high/low ratio increase compared to WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, below). Since both Lck bands were equally detected by the anti-phosphoTyr394 and the anti-phosphoTyr505 antibodies, we investigated if the differences in both Lck bands could be due to phosphorylation at the Ser59 site. By probing whole cell lysates of WT Jurkat cells with the anti-phospho-Lck(Ser59) antibody, we found that phosphorylation at that site peaked at 5 minutes after stimulation with anti-CD3 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Such induced phosphorylation was blocked in the presence of the MEK inhibitor UO126, thus proving that Ser59 phosphorylation was promoted by ERK when activated by MEK. Interestingly, the mobility of the Lck band detected by the anti-phospho-Lck(Ser59) antibody corresponded to the high-molecular weight form. Consequently, we investigated if the G169A mutation reduced the phosphorylation of Lck at Ser59 compared to WT Jurkat cells and found that this was the case (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The same blotting membrane was reprobed with the anti-phosphoTyr394 antibody, which confirmed that the high-molecular weight Lck band corresponded to the form phosphorylated at Ser59 and that such phosphorylation was inhibited in cells bearing the G169A mutation. Together, these results show that the G169A mutation in CD3ε inhibits the activation of early downstream effectors of TCR signaling, including Zap70, LAT, and ERK, and that it has a selective inhibitory effect on the feedback loop of ERK on Lck phosphorylation at Ser59.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe G169A mutation impairs the outside-in transmission of conformational changes in the TCR, preventing the exposure of the PRS in CD3ε.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo elucidate a possible mechanism for the deficient phosphorylation of specific tyrosine residues in CD3ε and CD3ζ when the TCR bears the G169A mutation, we investigated if this mutation impaired the transmission of the conformational change from the extracellular domains of the TCR to the PRS of CD3ε. To this end, we performed a pull-down assay on detergent cell lysates of Jurkat WT and G169A cells with beads coated with GST protein fused to the SH3.1 domain of Nck1, as schematized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. In WT cells, stimulation with anti-CD3 for 1 minute was already sufficient to induce the binding of the triggered TCR to the SH3.1 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, uncropped blots are in the Supplementary Information). This induced binding was observed by Western blotting with the three antibodies used, specific for CD3ζ, CD3γ, and TCRα, indicating that the entire TCR shifted from a non-competent Resting state to a competent Active state. Compared with a WT TCR, the G169A mutation impaired the anti-CD3-induced binding of the TCR to the SH3.1 domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), suggesting that the G169A mutation impaired the transmission of the conformational change along the cytoplasmic tail of CD3ε.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite the inducible binding of the entire TCR to GST-SH3.1, some binding of CD3ζ, but not of the other subunits, was detected at time 0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In another GST-SH3.1 pull-down experiment, the blotting membrane was incubated with the anti-phospho-CD3ζ(Tyr142) antibody. This showed some binding to the beads of the low-molecular weight form of CD3ζ with low phosphorylation even in lysates from resting cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). However, the binding of the high-molecular weight form of CD3ζ was exclusively detected after stimulation for one minute or longer, suggesting that the active TCR binding to the SH3.1 domain is fully tyrosine phosphorylated. The G169A mutation impaired such induced binding of the high-molecular weight, fully phosphorylated, form of CD3ζ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) and suggests that adoption of the Active conformation by the TCR, including the cytoplasmic tail of CD3ε, is required for full phosphorylation of CD3ζ.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eUnlike membrane tyrosine kinase receptors, the TCR has no intrinsic enzymatic activity and relies on the recruitment of cytoplasmic kinases that act in a concerted manner to phosphorylate tyrosine residues in the cytoplasmic tails of TCR subunits, and these phosphorylated tyrosines become docking sites for the recruitment of effector proteins. Even with this simplistic interpretation of the earliest mechanisms of TCR signaling, the TCR does not appear to be a \"passive\" receptor that is phosphorylated and dephosphorylated in response to what other membrane and cytoplasmic proteins determine. One of the arguments that the TCR is not just an on/off switch is the diversity of signaling subunits, cytoplasmic tails and ITAMs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The cytoplasmic tails of the CD3γ, CD3δ, CD3ε and CD3ζ subunits have been conserved since the emergence of the first jawed vertebrates, and this conservation does not only concern the tyrosine residues conforming to the ITAMs, but also extends upstream, downstream and to the inter-tyrosine sequences. A comparison of all the sequences of the cytoplasmic tails of CD3ε revealed that the most conserved motif was the PRS, which is involved in the recruitment of Nck, followed by the central tyrosine residues of the ITAM and other motifs previously described: the BRS\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and the RK motifs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In this paper, we have made point mutations affecting conserved residues of these novel motifs and found that all of them impaired the signaling capacity of the TCR, resulting in the release of IL-2. We focused our attention on an uncharacterized di-glycine motif located between the BRS and the PRS. A conservative mutation of one of these residues, Gly169, to alanine, resulted in the greatest reduction in IL-2 release, as well as reductions in the expression of the activation markers CD69 and CD25. As to what might be the root cause of the signaling defect in the TCR, we have found that the G169A mutation impairs the induced exposure of the PRS for Nck binding, suggesting that Gly169, and probably Gly170, are required for the outside-in transfer of the anti-CD3-induced conformational change in the TCR all the way from the transmembrane domain to the PRS. It has been proposed that the BRS in CD3ε is responsible for anchoring the cytoplasmic tail of the resting TCR to the inner leaflet of the membrane lipid bilayer and that TCR activation releases this anchoring\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. According to this model, the di-glycine motif could be important for information transfer to the motifs downstream of the BRS, including the PRS and the ITAM. Although we do not have experimental confirmation, our running hypothesis is that the G169A mutations have a structural impact on the cytoplasmic tail of CD3ε, rather than Gly169 being directly involved in the binding of an effector protein. Glycine residues are, after proline residues, the lowest amino acids on a scale of propensity to be found in α-helixes, whereas alanine residues are at the top\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Gly169 is preceded by an alanine, so it could be imagined that the G169A mutation allows the formation of half a turn of an α-helix, making the otherwise unstructured region adopt a structure less prone to transmitting conformational information from the N-terminus to the C-terminus in the cytoplasmic tail.\u003c/p\u003e \u003cp\u003eOne of the most striking consequences of the G169A mutation was the differential effect on the phosphorylation of specific tyrosine residues within the ITAMs of CD3ε and CD3ζ. The deficient phosphorylation of Tyr166 (N-terminal tyrosine) in the ITAM of CD3ε may be a consequence of impaired transduction of the conformational change, resulting in reduced exposure of the ITAM to phosphorylation by Lck. Interestingly, the effect on CD3ζ phosphorylation depended on the specific tyrosine residues, i.e. Tyr111 (ITAM ζb) and Tyr142 (ITAM ζc) did not seem to be affected. However, phosphorylation of Tyr83 (ITAM ζa) was inhibited in the presence of the G169A mutation. In our Western blots under non-reducing conditions, phospho-CD3ζ appeared as two distinct bands of low and high molecular weight, which could represent the dimers of the p21 and p23 forms of CD3ζ that have been studied for years\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Of note, a hierarchy of ITAM tyrosine phosphorylation has been proposed in which tyrosine residues in ITAMs ζb and ζc are first phosphorylated to form the p21 form, with ITAM ζa being the last to be phosphorylated to form the fully phosphorylated p23 form\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the presence of the G169A mutation, we detected an impaired conversion of the low-phosphorylated to the high-phosphorylated form of CD3ζ, and this coincides with a poor phosphorylation of Tyr83, which we detected only in the fully phosphorylated form. These data suggest that the G169A mutation prevents the phosphorylation of ITAM ζa and, consequently, the full phosphorylation of CD3ζ. How does the G169A mutation selectively affect the phosphorylation of ITAM ζa? One clue could be the RK motif located downstream of the first tyrosine (Tyr166) of CD3ε that has been implicated in TCR-triggered recruitment of Lck\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In that paper, an effect of a double mutation (RKAA) on CD3ζ phosphorylation was described, although no differential effect on specific tyrosine residues was reported. On the other hand, it has also been shown that the BRS in CD3ε is involved in the recruitment of Lck\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The di-glycine motif described in this work is placed between the BRS and the RK motifs, therefore, a working model (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee) could be that G169, and the di-glycine motif, is necessary to interconnect the two sequence features in CD3ε involved in Lck recruitment, perhaps by allowing the cytoplasmic tail of CD3ε to have the sufficient flexibility to interconnect both features. Our data suggest that the di-glycine motif upstream of the PRS is required to recruit Lck, being this recruitment necessary for the phosphorylation of the ITAM ζa and full CD3ζ phosphorylation.\u003c/p\u003e \u003cp\u003eAnother consequence of the G169A mutation is the impairment of the phosphorylation of Lck at Ser59 by ERK in a feedback loop. However, the consequences of such impairment of Ser59 phosphorylation are not yet fully understood due to controversy surrounding its positive or negative role. For instance, initial observations suggest that Ser59 phosphorylation reduces Lck activity\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and inhibits TCR signaling in Jurkat T cells expressing a phosphomimetic S59E Lck mutant\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Conversely, another study has shown that phosphorylation of Ser59 interferes with the interaction between Lck and the phosphatase SHP-1, preventing dephosphorylation of Tyr394 and subsequent Lck inactivation in thymocytes\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, blockade of the Erk1/2-Lck feedback loop, and hence Ser59 phosphorylation, rapidly terminates proximal TCR signaling\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In any case, the effect of the G169A mutation on Ser59 phosphorylation appears to be secondary to the effect on ITAM ζa phosphorylation, as the latter occurs with faster kinetics (a peak at 1 min versus a peak at 5 min of stimulation).\u003c/p\u003e \u003cp\u003eRegardless, what is ultimately responsible for the defective T cell activation observed in late events (IL-2 release, CD69 and CD25 expression), our findings on the effects of the G169A mutation in CD3ε suggest that direct TCR signaling is a finely-tuned, stepwise process. This process involves an outside-in and N-terminal-to-C-terminal transfer of information within CD3ε, leading to selective impacts on specific residues not only of CD3ε but also of CD3ζ, and influencing the activity and phosphorylation of the associated tyrosine kinase Lck. These data support conformational models of intra-TCR signaling and challenge models based on simple molecular exclusion. Further research is needed to understand the structural significance of the glycine-to-alanine mutation at position 169.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cb\u003eCells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe human T-cell Jurkat line was maintained in complete RPMI 1640 supplemented with 5% fetal bovine serum (FBS, Sigma).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmids.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe pGEX-4T1 derivative GST-SH3.1 containing the amino-terminal SH3 domain of Nck1 was kindly provided by R. Geha (Children\u0026rsquo;s Hospital, Harvard Medical School, Boston, MA). Point mutations in human CD3ε were made using the QuickChange-XL (Stragene). The FLAG-epitope was added to the C-terminus and the cDNA encoding human CD3ε was cloned into pSRα The GFP-expressing HIV vector pHRSIN-WPRE\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e was provided by Dr. J.A. Pintor (CABIMER-CSIC, Sevilla) wherein murine CD3ε cDNA replaced GFP to construct pHRSIN-mCD3ε.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of CD3ε knockout Jurkat cells by CRISPR/Cas9\u003c/h2\u003e \u003cp\u003eTo inactivate the endogenous \u003cem\u003eCd3e\u003c/em\u003e gene in Jurkat cells, we utilized the CRISPR/Cas9 system as described by Ran et al \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Briefly, sgRNAs specific to the target gene were designed using an online CRISPR Design tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tools.genome-engineering.org\u003c/span\u003e\u003cspan address=\"http://tools.genome-engineering.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). One set of sgRNAs was used targeting EXON 2 of the \u003cem\u003eCd3e\u003c/em\u003e gene (Suppl. Table\u0026nbsp;1). DNA oligos encoding the top and bottom sgRNA strands were hybridized and ligated into the pSpCas9(BB)-2A-GFP (PX458) vector. These vectors were then electroporated in circular form into Jurkat cells. Forty-eight hours post-electroporation, single cell sorting was performed to isolate individual CD3εKO Jurkat clones that were GFP\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e\u0026minus;\u003c/sup\u003e by flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of mutant CD3ε G169A Jurkat cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, vectors containing CD3ε mutations were generated using direct mutation PCR with primers listed in Supplementary Table\u0026nbsp;1. Once the mutations were obtained, the PSRalpha vector containing mutated CD3ε was electroporated into Jurkat CD3εKO cells. CD3ε mutant clones expressing TCR levels comparable to WT Jurkat cells were selected (Suppl. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibodies and other reagents.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe rabbit anti-CD3ζ antiserum 488 has been described previously\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The other antibodies used were purchased as follows: hybridomas producing the anti-human CD3 mAb OKT3 from ATCC; the anti-phospho-CD3ζ (pTyr83), Anti-phospho-CD3ζ (pTyr142), anti-phospho-CD3ζ (pTyr111), anti-HA (12CA5) from Sigma; HRP Mouse Anti-Phosphotyrosine PY20 from BD biosciences; rabbit anti-mouse pERK (T202/Y204), anti-phospho Zap70 (Y319)/Syk(Y352), anti- phospho-Lck (Tyr505), anti-phospho-Lck (Y394), anti-phospho LAT (Y132), anti-phospho Src family (Y416) from Cell Signalling; anti-GST peroxidase conjugated from Rockland Immunochemicals; anti-phospho Lck (Y394) from RB Systems; anti-phospho Lck (Ser59), anti-CD3γ and anti-TCRα from Thermo Fisher scientific.\u003c/p\u003e \u003cp\u003e \u003cb\u003eT cell stimulation and flow cytometry.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor CD69 and CD25 expression, 2x10\u003csup\u003e5\u003c/sup\u003e cells were analyzed 24 h after stimulation with different concentrations of anti-CD3 OKT3 or PMA and ionomycin. Anti-CD28 (0.1 \u0026micro;g/ml) was added for all anti-CD3 concentrations. After stimulation, cells were washed in PBS\u0026thinsp;+\u0026thinsp;2%FBS and stained with anti-human CD69-FITC and CD25-APC (Invitrogen) antibodies for 30 min at 0\u0026ordm;C. Cells were then washed twice and analyzed by flow cytometry (FACS Canto II Becton Dickinson) and FlowJo software. Dead cells were excluded in all analyses by using DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eT cell stimulation and Western blotting\u003c/h2\u003e \u003cp\u003eJurkat cells were resuspended in RPMI plus 20mM Hepes and left under starving conditions overnight. The MEK inhibitor U0126 (20 \u0026micro;M) was added when indicated. Cells were stimulated at different time points with anti-CD3 OKT3 antibody. After stimulation, cells were lysed in Brij96 lysis buffer containing protease and phosphatase inhibitors (0.33% Brij96, 140mM NaCl, 20mM tris-HCl (pH7.8), 10mM iodoacetamide, 1mM PMSF (phenylmethylsulfonyl fuoride), leupeptin (1\u0026micro;g/ml), aprotinin (1\u0026micro;g/ml), 1mM sodium orthovanadate, and 20mM sodium fuoride). For western blotting, the samples were resolved by SDS\u0026ndash;polyacrylamide gel electrophoresis (PAGE). The recovered proteins were then transferred to a nitrocelullose membrane that was probed with different antibodies. Finally, the membrane was incubated with streptavidin-conjugated horseradish peroxidase (Southern Bio-technology) and developed by ECL (Pierce).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation\u003c/h2\u003e \u003cp\u003eFor imunoprecipitation, whole-cell lysates of the stimulated cells were incubated with anti-CD3 (OKT3) antibodies bound to protein G-Sepharose beads. The immunoprecipitated material was washed 5 times in Brij96 lysis buffer and finally resuspended in 20 \u0026micro;l of Laemmli sample buffer. Samples were resolved by SDS-PAGE and immunoblotting was carried out for the identification of total membrane proteins associated with OKT3.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePull-down assay.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFor the GST-Nck pull-down assay, cell lysates were incubated with purified recombinant protein consisting of glutathione-S-transferase (GST) fused to the SH3.1 domain of human Nck1 or glutathione-S-transferase (GST) at 4\u0026ordm;C for 2 hours. After washing 5 times, the pull down beads were boiled in Laemmli sample buffer and subjected to SDS-PAGE and then transferred by Western blot to a nitrocellulose membrane (Bio-Rad) which was probed with different antibodies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection of human IL-2 by ELISA\u003c/h2\u003e \u003cp\u003eIL-2 detection was performed using DuoSet\u0026reg; ELISA development system kits (R\u0026amp;D Systems, Minneapolis, MN, USA) according to the manufacturer\u0026rsquo;s protocol. The experiments were carried out in triplicates and in a 96-well ELISA plate and washes were carried out using Wash buffer (PBS with 0,05% Tween\u0026reg;-20) three times per wash. The plate was coated with α-hIL-2 capture antibody (4 \u0026micro;g/ml), sealed and incubated overnight. The coated antibody was discarded and the plate was washed. The plate was then incubated with Block buffer (PBS with 1% bovine serum albumin (BSA), 0,22 \u0026micro;m filtered) for 1 hour at room temperature. After washing, the 100 \u0026micro;l/well of the samples were plated. Dilutions of R-hIL-2 standards (as recommended by the manufacturer) were prepared in Reagent diluent (Tris-buffered saline (TBS) with 0,1% BSA, 0,05% Tween\u0026reg;-20, pH 7,2\u0026ndash;7,4; 0,22 \u0026micro;m filtered) and made in duplicates for generation of standard curve. Plates were sealed and incubated 2 hours at room temperature. The plate was washed and the detection antibodies g-α-hIL-2-biotin (BIO) (100 ng/ml) diluted in Reagent diluent were incubated for 2 hours at room temperature. The plate was washed and incubated with streptavidin-horseradish peroxidase (STV-HRP) for 20 min in the dark at room temperature. Substrate solution (1:1 mix of 3,3',5,5'-tetramethylbenzidine (TMB) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) was added, after washing, and incubated up to 20 min in the dark at room temperature. 50 \u0026micro;l/well Stop solution (2N H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was added to stop the reaction and optical density was measured using iMark\u0026trade; microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA) at 450nm with subtractive correction at 595nm.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRT carried out most of the experiments; ERB and NMB generated mutations, prepared constructs and selected clones; AL carried out some of the immunoblottings; BA conceived the project, directed the research and wrote the manuscript, which was edited and revised by RT, ERB and AL.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are indebted to Valentina Blanco and Tania G\u0026oacute;mez for their expert technical assistance. This work was supported by Grant PID2022-136745OB-I00 funded by AEI/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.13039/501100011033\u003c/span\u003e\u003cspan address=\"10.13039/501100011033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e and, by the \u0026ldquo;European Union NextGenerationEU/PRTR\u0026rdquo;, and by Grant P2022/BMD7209 (INTEGRAMUNE-CM) from the \u0026lsquo;Comunidad de Madrid\u0026rsquo;.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the conclusions of this article are presented within article and its additional files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeddoe, T., Chen, Z., Clements, C.S., Ely, L.K., Bushell, S.R., Vivian, J.P., Kjer-Nielsen, L., Pang, S.S., Dunstone, M.A., Liu, Y.C., et al. (2009). 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Eur J Immunol \u003cem\u003e28\u003c/em\u003e, 12\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"T cell receptor, Allostery, CD3ε, Nck binding, Lck recruitment, CD3ζ phosphorylation","lastPublishedDoi":"10.21203/rs.3.rs-4594242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4594242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHow the T cell receptor (TCR) recognizes its antigen ligand and transmits this information to the cytoplasm for T cell activation is still a matter of debate. A large body of experimental data suggests that the TCR undergoes allosteric changes upon ligand binding that are responsible for the outside-in transfer of information. One of the hallmarks of TCR allostery is the induced exposure of a proline-rich sequence (PRS) in the cytoplasmic tail of the CD3ε subunit, enabling binding to the cytoplasmic adaptor protein Nck. We show here that a glycine-to-alanine mutation (G169A) in a conserved di-glycine motif upstream of the PRS impairs TCR binding to Nck upon stimulation with an activating antibody. Furthermore, the mutation hinders CD3ε tyrosine phosphorylation and, interestingly, selectively affects CD3ζ subunit phosphorylation at Tyr83. In addition, the G169A mutation impairs the phosphorylation of Lck at Ser59. Taken together, these data support the concept of TCR allostery and highlight the existence of a regulated sequence of interactions between TCR subunits and associated effector proteins.\u003c/p\u003e","manuscriptTitle":"A di-glycine motif in the cytoplasmic tail of CD3ε required for transmission of allosteric changes in the TCR and full CD3ζ phosphorylation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 16:22:29","doi":"10.21203/rs.3.rs-4594242/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9371743-201f-4aad-b9f4-607af1843b8f","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34749041,"name":"Biological sciences/Immunology/Signal transduction"},{"id":34749042,"name":"Biological sciences/Immunology/Lymphocytes/T cells/T cell receptor"}],"tags":[],"updatedAt":"2024-07-23T04:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 16:22:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4594242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4594242","identity":"rs-4594242","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}