The Y105 phosphosite between the SH3.1 and SH3.2 domains of Nck1 regulates its binding to CD3ϵ

Summary

In αβ T cells, ligand binding to T cell receptor (TCR) triggers conformational changes at CD3ε subunits to expose the proline-rich sequence (PRS), which binds to the non-catalytic region of tyrosine kinase (Nck) via its Src homology 3 (SH3).1 domain. Recruitment of Nck to CD3ε initiates phosphorylation of various signaling proteins that activate T cells. However, the mechanisms by which Nck associates with and dissociates from the TCR remain poorly understood. This study identifies the tyrosine 105 (Y105) phosphosite between the SH3.1 and SH3.2 domains of Nck1 that regulates the Nck1-CD3ϵ association. Notably, a Y105 mutation resulted in prolonged association of Nck1 with TCR, impaired the phosphorylation of CD3ε, TCRζ, ζ-chain-associated protein kinase 70, and extracellular signal-regulated kinase 1/2, and abrogated the recruitment of lymphocyte-specific kinase (Lck) to TCR. These findings revealed the function of Y105 as a molecular switch that controls the assembly and disassembly of Nck1 and CD3ε.

Graphical abstract

Highlights

• •The conserved tyrosine residue Y105 of Nck1 is phosphorylated upon T cell receptor stimulation
• •Tyrosine 105 of Nck1 serves as a molecular switch for Nck1–CD3ε assembly and disassembly
• •Tyrosine 105 of Nck1 is required for T cell activation
• •Lck is required for Nck1 phosphorylation

Cell biology; Immunology; Molecular biology

Introduction

Signals transmitted from surface receptors to downstream signaling molecules are required for T cell activation and effector functions in adaptive immunity. T cells express diverse T cell receptors (TCRs) on the cell surface to specifically recognize antigenic peptide-loaded major histocompatibility complex molecules in antigen-presenting cells. TCR is composed of the variable antigen-binding TCRαβ heterodimer that is noncovalently associated with the non-variable CD3εγ, CD3εδ, and ζζ signal transduction subunits to form the TCR-CD3 complex.¹ The cytoplasmic tail of each CD3 subunit contains a single immunoreceptor tyrosine (Tyr/Y)-based activation motif (ITAM), whereas the ζ subunit has three ITAMs.² The ITAMs are phosphorylated by the lymphocyte-specific kinase (Lck) after TCR engagement. Although the consequence of TCR triggering and subsequent initiation of signal transduction remains unclear, a conformational change of CD3ε into the active conformation upon TCR triggering has been proposed.^3,4,5,6 The cytoplasmic tail of CD3ε has various unique motifs for binding with its binding partners to initiate TCR signaling.⁷ From the N to C terminus, CD3ε contains a basic amino acid-rich sequence (BRS), a proline-rich sequence (PRS), and a receptor kinase (RK) motif or ITAMs. These motifs are accessible to their binding partners after the TCR-induced conformational changes of CD3ε into an active conformation. In the active conformation of CD3ε, the BRS and ITAM motifs are responsible for interacting with the unique region and the SH3 domains of Lck, respectively,⁸ whereas the CD3ε PRS is the binding site for the non-catalytic region of Tyr kinase (Nck).³

Nck is an adaptor protein that lacks intrinsic catalytic activity. However, Nck acts as a linker to transmit the signal from the TCR-CD3 complex to downstream signaling events for actin reorganisation.^7,9,10,11 The following two highly conserved members of the Nck family that share 68% amino acid identity have been identified in humans: Nck1/Nckα and Nck2/Nckβ (also known as Grb4).^10,12,13 Both Nck proteins comprise three SH3 domains (SH3.1, SH3.2, and SH3.3) at the N-terminal and one SH2 domain at the C-terminal.¹⁰ Nck SH3 motifs provide the site for interacting with PRS-bearing proteins, whereas the Nck SH2 motif facilitates its binding to phosphotyrosine (pTyr)-containing proteins.⁹ Apart from its effect on actin polymerization, Nck is also involved in the regulation of TCR-induced signals leading to T cell activation. In the light of T cell activation, Nck1, rather than Nck2, was found to play a major role in TCR signaling.¹⁴ Moreover, the Nck SH3.1 domain is recruited to bind to the CD3ε PRS after TCR-triggered conformational changes of CD3.³ Initially, the recruitment of Nck to CD3ε occurs earlier and independently of Tyr phosphorylation.³ In conjunction with the binding of the Nck SH3.1 domain and the CD3ε PRS, the partially phosphorylated CD3ε is also required for efficient Nck binding to CD3ε.¹⁵ The recruitment of Nck to CD3ε is necessary for phosphorylation of CD3 ITAMs, and ζ chain-associated protein 70 (ZAP70) initiates signal progression.¹⁶ Furthermore, Nck has been found to be essential for Lck recruitment to the CD3ε ITAM motif, where Nck and Lck cooperate to induce optimal TCR signaling.⁸

The Nck-CD3ε interaction peaked at 5 min and declined at 10 min after TCR stimulation.¹⁵ However, the mechanisms underlying the association and disassociation between Nck and CD3ϵ remain largely unknown. Moreover, the molecular structure of the Nck1 protein revealed that the linker between the Nck1 SH3.1 and SH3.2 motifs harbors a Tyr residue at position 105 (Y105), which has no identified function in T cells. However, in human endothelial cells, the Y105 residue of Nck1 is phosphorylated by Abelson murine leukemia viral oncogene homolog 1 (c-ABL) to down-modulate p38.¹⁷ In addition, the genetically encoded pTyr analog at the Y55 position in the Nck1 SH3.1 domain completely abolished Nck1 binding to CD3ε.¹⁸ Considering the aforementioned evidence, this study proposed that the Y105 residue of Nck1 may be involved in regulation of Nck1 association with and disassociation from CD3ε in Jurkat T cells. Thus, the study aimed to assess the potential role of Y105, which is located between the SH3.1 and SH3.2 domains of Nck1, in facilitating the Nck1-CD3ε interaction.

The study revealed that the conserved Y105 residue could be phosphorylated. Moreover, the duration of Nck1 disassembly from CD3ε correlated with the level of Nck1 phosphorylation, in which full Nck1 phosphorylation terminated its association with CD3ε. Furthermore, inhibition of Y105 phosphorylation by mutating the residue impeded the disassociation of Nck1 from CD3ε in conjunction with the defect in TCR-proximal and TCR-distal signaling progression. Furthermore, Nck1 was directly phosphorylated by the Src-family kinase Lck, and the mutation of Y105 also disrupted Nck1-Lck binding, resulting in impaired CD3ϵ phosphorylation and CD69 expression.

The phosphorylation of Nck is restricted primarily to serine (Ser) and threonine residues.¹⁹ Using mass spectrometry (MS), seven different pTyr residues have been identified in Nck.²⁰ Among these Tyr residues, three Tyr residues (Nck1 Y246 located within the SH3.3 domains, and Nck2 Y55 and Y251 located within the SH3.1 and SH3.3 domains, respectively) were confirmed to be phosphorylated.²⁰ Since this Nck Y105 is conserved among species, it paves the way to explore its role in T cell signal transduction. Alignment of the amino acid sequences between Nck1 and Nck2 proteins revealed the conserved consensus sequences of Y105 among species as I/L/V-Y-X_1-5-P/F.¹⁷

Results

Nck contains an evolutionarily conserved Tyr in the linker between the SH3.1 and SH3.2 domains

To assess the Tyr phosphorylation of Nck1, Jurkat T cells were stimulated with the anti-TCR antibody C305 or left unstimulated. Nck1 was immunoprecipitated and immunoblotting was performed with the anti-pTyr antibody 4G10. Time course analysis showed that Nck1 was phosphorylated after 5 min and increased at 10 min (Figure 1A). These data indicated that TCR stimulation induces Nck1 phosphorylation of Tyr. This is in line with previous reports showing Nck phosphorylation at the Ser, Thr, and Tyr residues.^18,19,20,21

Figure 1.

Y105 of Nck1 is conserved and Nck1 can be phosphorylated upon Jurkat T cell activation

(A) Nck1 phosphorylation increases after TCR stimulation in Jurkat cells. Cells were either unstimulated or stimulated with anti-TCR antibody (C305) at 37°C for various times. Nck1 was immunoprecipitated, followed by SDS-PAGE and immunoblotting with anti-Nck1 and anti-phosphotyrosine antibodies. Phospho-Nck1 levels normalized to total Nck1 were quantified using ImageJ. Results represent three independent experiments (n = 3), shown as mean ± SEM.

(B) Nck1 contains three N-terminal SH3 domains (SH3.1, SH3.2, SH3.3) and a C-terminal SH2 domain. The Y105 phosphosite lies between SH3.1 and SH3.2.

(C) Sequence alignment of amino acids 95–118 from human Nck1 and Nck2 across species shows conserved Y105, within the consensus sequence E(D)RL(I)YDLNXPA. Abbreviations: Y105, tyrosine 105; Nck, non-catalytic region of tyrosine kinase; TCR, T cell receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM, standard error of mean; SH2, Src homology 2.

Y105 of Nck1 is located within the linker between the SH3.1 and SH3.2 domains (Figure 1B) and is evolutionarily conserved from humans to cartilaginous fish in Nck1 and Nck2 (Figure 1C). This was true for Nck1 and Nck2 (Figure 1C). Furthermore, the surrounding amino acids are also conserved, suggesting an important functional role. Thus, whether Y105 is phosphorylated and whether it plays a role in signal transduction in T cells was explored in this study.

Y105 is phosphorylated in Jurkat T cells upon TCR triggering

Nck1 plays a more dominant role than Nck2 in T cell activation.¹⁴ Therefore, this study focused on the Th function of the Y105 residue of Nck1. The NCK1 gene was knocked out using CRISPR-Cas9 to generate Nck1-knockout (N1KO) Jurkat T cells. As expected, Nck1 expression was completely abolished in N1KO Jurkat T cells compared with that in parental Jurkat T cells (Figure 2A). Furthermore, Nck2 expression remained unaltered in N1KO Jurkat T cells.

Figure 2.

TCR engagement induces Nck1 Y105 phosphorylation

(A) Nck1 was knocked out (N1KO) in Jurkat T cells using CRISPR-Cas9 with Nck1-specific guide RNA. Transfected cells were selected with puromycin for seven days, lysed, and analyzed by western blot. Band intensities of Nck1 and Nck2 were normalized to GAPDH. Data represent three independent experiments (n = 3) (mean ± SEM).

(B) Nck1 knockout reduced CD3ε phosphorylation. Parental and N1KO cells were untreated or treated with PV. Lysates were immunoprecipitated with anti-phosphotyrosine antibodies (4G10) and immunoblotted with indicated antibodies. Band intensities were quantified relative to IgG (B, right). Data are mean ± SEM from three experiments (n = 3).

(C) Schematic of WT-Nck1 and Y105F mutant constructs, with a FLAG tag at the N terminus. Tyrosine 105 between SH3.1 and SH3.2 was mutated to phenylalanine (Y105F).

(D) WT-Nck1 and Y105F-Nck1 proteins were re-expressed in Nck1-deficient Jurkat cells and confirmed by immunoblot (n = 2).

(E) CD3 surface expression in parental, Nck1-KO, WT-Nck1-, and Y105F-Nck1-reconstituted cells was measured by flow cytometry (PerCP-anti-CD3) and shown as mean fluorescence intensity (mean ± SEM, n = 3).

(F) Phosphorylation of Nck1 was impaired in Y105F mutant cells. After stimulation, Nck1 was immunoprecipitated and blotted for phospho-Nck1. Quantification showed reduced phosphorylation in Y105F (mean ± SEM, n = 3).

(G) Mass spectrometry of primary CD4 T cells and Jurkat cells treated with anti-CD3 (OKT3) showed Y105 phosphorylation upon stimulation, absent in untreated cells (n = 2). Statistical analysis: unpaired Student’s t test, ns = not significant, ∗∗p < 0.01, ∗∗∗p < 0.001. Abbreviations: Nck, non-catalytic region of tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; IgG, immunoglobulin G; Y105, tyrosine residues at position 105; PerCP, peridinin-chlorophyll-protein.

To maximize tyrosine phosphorylation of Nck1, pervanadate (PV), a pan phosphatase inhibitor, was used. PV has been reported to induce phosphorylation of various TCR-proximal signaling proteins as found in TCR stimulation.²² N1KO and parental Jurkat cells were treated with the phosphatase inhibitor PV or left unstimulated. Tyr phosphorylation of Nck1 was investigated by immunoprecipitation with the anti-pTyr antibody 4G10, followed by the detection of Nck1 by immunoblotting. PV induced Tyr phosphorylation of Nck1 in parental Jurkat T cells but not in N1KO cells (Figure 2B). In line with previous findings,²³ phosphorylation of CD3ε was markedly reduced in PV-treated N1KO cells (Figure 2B). Thus, this result confirms that Nck1 can be phosphorylated.

To explore the potential function of the Y105 residue in Nck1, N1KO cells were reconstituted with Flag-tagged wild-type Nck1 (WT-Nck1) or mutated Nck1 (Y105F-Nck1), in which Y105 was replaced with phenylalanine (Figure 2C). Reconstitution of N1KO cells with either Flag-tagged WT-Nck1 or Y105F-Nck1 restored the expression of Nck1 to levels comparable with those of the endogenous Nck1 protein from parental Jurkat T cells (Figure 2D). As previously mentioned, Nck2 expression remained unaltered. In addition, similar levels of TCR surface expression were observed in parental, N1KO, WT-Nck1, and Y105F-Nck1 cells (Figure 2E).

To test whether Y105 was phosphorylated in Jurkat T cells, the N1KO cells expressing WT-Nck1 or Y105F-Nck1 were stimulated with anti-TCR antibody C305, Nck1 was immunoprecipitated, and Tyr phosphorylation was assessed using immunoblotting with the anti-pTyr antibody 4G10 (Figure 2F). WT-Nck1 was phosphorylated; however, this was not the case for Y105F-Nck1. This suggests that upon TCR triggering, Y105 is the main Tyr phosphorylation site on Nck1.

To directly test the phosphorylation of Y105, Jurkat cells were stimulated with the anti-CD3 antibody OKT3 or left unstimulated and a tandem mass spectrometry (MS/MS) analysis was performed. The resulting peptide fragmentation spectra confirmed phosphorylation of Nck1 at Y105 following TCR stimulation (Figure 2G, left). To verify this observation in primary cells, human CD4⁺ T cells were isolated, stimulated with OKT3 or left unstimulated. Consistent with the results in Jurkat T cells, MS/MS analysis also revealed phosphorylation at Y105 of Nck1 in primary CD4⁺ T cells upon TCR activation (Figure 2G). To determine whether the anti-phosphotyrosine antibody (4G10) could detect phosphorylated Nck1 at Y105 in primary CD4⁺ T cells, cells were stimulated with OKT3, followed by Nck1 immunoprecipitation. Indeed, the Y105-phosphorylated form of Nck1 was also detected in primary CD4⁺ T cells (Figure S1). Collectively, these results demonstrate that Nck1 is phosphorylated at Y105 in response to TCR engagement in both Jurkat cells and primary CD4⁺ T cells.

Y105 is required for optimal phosphorylation of the TCR and downstream effector proteins in Jurkat T cells

The effect of the Nck1-Y105F mutation on TCR signaling was evaluated. Compared with N1KO cells expressing WT-Nck1, anti-pTyr immunoprecipitation showed that the Y105F mutation reduced Tyr phosphorylation of Nck1 after TCR engagement (Figure 3A). As previously observed²³ and as shown in Figure 2B, Nck1 was required for efficient CD3ε phosphorylation. Importantly, Y105F-Nck1 could not substitute for WT-Nck1 in the TCR-induced phosphorylation of Tyr1 in CD3ε (pY1-ε) (Figure 3A). Thus, Y105 is crucially involved in the function of Nck1 mediating CD3ε phosphorylation.

Figure 3.

Inhibition of Y105 phosphorylation in Nck1 by mutating this tyrosine residue that is associated with reduction of tyrosine phosphorylation on TCR and TCR-proximal signaling proteins in Jurkat T cells

(A) Mutation of Nck1 Y105 reduced phosphorylation of Nck1 and CD3ε. N1KO, WT, and Y105F Nck1-reconstituted Jurkat cells were untreated or stimulated with anti-TCR antibody (C305). Lysates were immunoprecipitated with anti-phosphotyrosine (4G10) antibodies and immunoblotted for indicated proteins. Representative blots and quantification of phospho-Nck1 and phospho-CD3ε normalized to IgG are shown (n = 3).

(B) Phosphorylation of CD3ζ was impaired in Y105F cells compared to WT, shown by immunoblot and quantification normalized to CD3ε (n = 3).

(C and D) Upon stimulation, WT and Y105F cells were immunoblotted for phospho-ZAP70 (C) and phospho-ERK1/2 (D). Quantification normalized to total protein is presented as mean ± SEM (n = 3). Statistical significance was assessed by unpaired student’s t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Abbreviations: Y105, tyrosine residues at position 105; Nck, non-catalytic region of tyrosine kinase; TCR, T cell receptor; IgG, immunoglobulin G; ZAP70, ζ chain-associated protein 70; ERK, extracellular signal-regulated kinase; SEM, standard error of the mean.

To test whether this function was restricted to CD3ε, or whether it also held true for the phosphorylation of TCR in general, CD3ζ was also examined. After TCR stimulation, CD3ζ phosphorylation in N1KO T cells expressing WT-Nck1 was detected as early as 1 min after stimulation, peaking at 5 min, and gradually decreasing at later time points (Figure 3B). Interestingly, N1KO cells expressing Y105F-Nck1 showed a marked decrease in CD3ζ phosphorylation.

Reduced TCR phosphorylation leads to reduced phosphorylation of ZAP70. Accordingly, this was the case in Y105F-Nck1 cells compared with that in WT-Nck1 cells (Figure 3C). To confirm the consequence of diminished ZAP70 activation in Y105F-Nck1 cells, the phosphorylation of Tyr 128 of SH2 domain-containing leukocyte protein (SLP)-76, which is phosphorylated by ZAP70 upon T cell activation, was monitored.²⁴ As anticipated, a decrease in SLP-76 phosphorylation in Y105F-Nck1 cells was observed compared to that in WT-Nck1 cells (Figure S2). To test whether the mutation in Y105 in Nck1 affected distal TCR signaling, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 was examined. In line with the proximal TCR signaling proteins, mutation of Nck1 at Y105 severely reduced the activation of ERK1/2 (Figure 3D).

The effect of Y105 mutation on CD69 expression was then analyzed. CD69 expression is normally used as a marker of early T cell activation.²⁵ Compared with that of WT-Nck1, Y105F-Nck1 considerably decreased CD69 expression (Figure 4A). The defect in T cell activation in Y105F-Nck1 cells was also confirmed by flow cytometry analysis of intracellular IL-2 production. A marked reduction of IL-2-positive cells in Y105F-Nck1 cells relative to the parental Jurkat and WT-Nck1 cells was seen (Figure 4B). This was similar to that observed in N1KO cells, corroborating that in Y105F-Nck1 cells TCR-induced events were strongly reduced. Given that Nck regulates TCR-induced actin rearrangement,¹⁰ it was hypothesized that Y105 may be involved in this process. Accordingly, TCR stimulation led to actin polymerization, showing an apical ring of F-actin in parental Jurkat T cells (Figure 4C). As expected, N1KO cells failed to reorganize actin after TCR activation. Expression of Y105F-Nck1 did not restore Nck1 KO, whereas WT-Nck1 did (Figure 4C). Altogether, these results reveal an indispensable role of Y105 in the function of Nck1 downstream of the TCR.

Figure 4.

Mutation of Y105 on Nck impairs CD69 expression and actin polymerization

(A) Mutation of Nck1 Y105 abolished CD69 expression after 18 h stimulation with anti-CD3/CD28 antibodies, measured by flow cytometry on CD3⁺ cells (n = 3).

(B) Y105F-Nck1 cells showed fewer IL-2 positive cells under the same stimulation, analyzed by intracellular staining and flow cytometry (n = 3).

(C) After 5 min anti-CD3 stimulation, polymerized F-actin was visualized with phalloidin-FITC and quantified (n = 3). Data are mean ± SEM; statistical significance by unpaired t test (ns, ∗∗p < 0.01, ∗∗∗p < 0.001). Abbreviations: Y105, tyrosine residues at position 105; Nck, non-catalytic region of tyrosine kinase; TCR, T cell receptor; IL, interleukin; APC, allophycocyanin; FITC, fluorescein isothiocyanate; SEM, standard error of the mean.

Phosphorylation of Nck1 Y105 increases the Nck1-CD3ϵ complex dissociation rate following TCR stimulation of Jurkat T cells

Next, the mechanism by which Nck1 promoted TCR phosphorylation was examined. Nck1 is recruited to the TCR upon TCR triggering.^3,15,23 This involves the SH3.1 domain of Nck1 binding to the PRS of CD3ε once the PRS becomes accessible upon ligand-binding to the TCR.²⁶ This is blocked by phosphorylation of Tyr1 in CD3ε which overlaps with the PRS.¹⁵

This study confirmed that early stimulation (5 min) with Nck1 was associated with TCR, which correlated with weak CD3ε phosphorylation (Figure 5A). Prolonged activation caused disassociation of Nck1 from CD3ε when CD3ε phosphorylation increased (Figure 5A). This finding suggested that the association of Nck1 with TCR required the partial phosphorylation of CD3ε (at Tyr2) and a full CD3ε phosphorylation (Tyr1 and Tyr2) led to the disassociation of Nck1 from CD3ε. These results were in agreement with previous findings¹⁵ that revealed that CD3ε double phosphorylation occurs in a stepwise manner.²⁷

Figure 5.

Inhibiting phosphorylation of Y105 in Nck1 inhibits its disassociation from CD3 molecules

(A) Nck1 is recruited to CD3ε early after TCR stimulation and dissociates later, shown by immunoprecipitation of Jurkat cells stimulated with anti-TCR antibody (C305) at various times. Lysates were immunoprecipitated with anti-CD3 and blotted for Nck1, phospho-CD3ε (pCD3ε), and CD3ζ. Band intensities were quantified and normalized to CD3ζ (mean ±SEM, n = 3).

(B) The in situ PLA detects close protein proximity (10–80 nm) via antibody-linked DNA amplification, visualized by fluorescence microscopy.

(C) PLA showed Nck1-CD3 interaction peaks at 5 min post-TCR stimulation and declines thereafter. Negative controls lacked one primary antibody. Quantification of PLA signals (red dots) is presented as mean ± SEM (n = 3).

(D and E) In cells expressing Y105F-Nck1, phosphorylation inhibition prevents Nck1 dissociation from CD3ε, demonstrated by sustained PLA signals (D) and immunoprecipitation (E) after stimulation. Quantification of band intensities (Nck1 normalized to pCD3ε) and PLA dots is shown (mean ±SEM, n = 3). Statistical significance was assessed by two-tailed student’s t test (∗p < 0.1, ∗∗∗∗p < 0.0001, ns = not significant). Abbreviations: Y105, tyrosine residues at position 105; Nck, non-catalytic region of tyrosine kinase; TCR, T cell receptor; PLA, proximity ligation assay; DAPI, 4′,6-diamidino-2-phenylindole; SEM, standard error of the mean.

Additionally, the proximity ligation assay (PLA) technique was employed to visualize the close proximity between two proteins in situ²⁸ in Nck1 and the cytoplasmic tail of CD3ε (Figure 5B). The presence of red fluorescent dots indicated Nck-CD3ε proximity. Consistent with the immunoblotting results, the TCR-induced proximity between Nck1 and CD3ε was prominently detectable after 5 min but sharply declined after 10 and 30 min of stimulation (Figure 5C).

The mechanisms underlying the association and dissociation of Nck and CD3ε have not yet been fully explored. To investigate whether phosphorylation of Y105 in Nck1 plays a role, PLA was used to visualize the Nck1-CD3ε interaction in N1KO, WT-Nck, and Y105F-Nck1 cells. After 5 min of stimulation, Nck1-CD3ε PLA dots were detected in both WT-Nck1 and Y105F-Nck1 cells as compared to the unstimulated condition (Figure 5D). Importantly, when cell activation was extended for 10 min the co-localization of Nck1 and CD3ε in WT-Nck was reduced, whereas this binding was not diminished in Y105F-Nck1 cells. This finding suggests that the phosphorylation of Nck1 Y105 is necessary to control the dissociation of Nck1 from CD3ε. Consistent with the PLA results, immunoprecipitation of CD3ε showed increased binding of Nck1 to CD3ε after 5 min of stimulation in WT-Nck1 and Y105F-Nck1 cells (Figure 5E). Upon prolonged activation for 10 min, the amount of Nck1 that co-immunoprecipitated with CD3ε was reduced in WT-Nck1 cells but not in Y105F-Nck1 cells. Taken together, these findings suggest a previously unidentified function of the conserved Y105 residue of Nck1 in regulating the dissociation of Nck1 from CD3ε after longer TCR stimulation.

Y105 of Nck1 supports the association of Lck with the TCR

Nck1 and Lck directly or indirectly bind to each other.^8,29 Hence, the possible involvement of Nck1 Y105 in the Nck1-Lck interaction was explored. To this end, Lck was immunoprecipitated in WT-Nck1 and Y105F-Nck1 expressing N1KO cells and Nck1 co-immunoprecipitation was performed. Binding of Nck1 to Lck was detected in WT-Nck cells after TCR stimulation for 5 min; however, this interaction tended to decrease after 10 min (Figure 6A). Interestingly, Y105F-Nck1 association with Lck was abolished at both stimulation time points. In contrast, anti-Nck1 immunoprecipitation showed that the mutation of Nck1 at Y105 abolished the binding between Nck1 and Lck upon TCR triggering (Figure 6B). As shown in Figure 3A, CD3ε was not phosphorylated in the Y105F-Nck1 cells but was phosphorylated in WT-Nck1 cells. This finding was consistent with the reduced phosphorylation of CD3ζ, ZAP70, and ERK1/2 in Y105F-Nck1 cells (Figures 3B–3D).

Figure 6.

Mutation of Y105 on Nck1 impairs Nck1 binding to Lck and CD3ε

(A and B) Y105 of Nck1 is essential for its interaction with Lck. WT and Y105F Nck1-reconstituted cells were unstimulated or stimulated with anti-TCR antibody (C305). Lysates were immunoprecipitated with anti-Lck (A) or anti-Nck1 (B) antibodies and immunoblotted for indicated proteins. Representative blots and quantification of Nck1 normalized to Lck (A) and Lck or CD3ε normalized to IgG (B) are shown ( mean ±SEM, n = 3).

(C) Y105F mutation impairs Lck recruitment to CD3ε. CD3 immunoprecipitation followed by immunoblotting for Lck, phosphorylated tyrosine (pεY1), and CD3ζ revealed reduced Lck association in mutant cells. Quantification normalized to CD3ζ is presented as mean ± SEM (n = 3). Statistical analysis by unpaired student’s t test (ns = not significant, ∗∗∗∗p < 0.0001). Abbreviations: Y105, tyrosine residues at position 105; Nck, non-catalytic region of tyrosine kinase; Lck, lymphocyte-specific kinase; TCR, T cell receptor; IgG, immunoglobulin G; SEM, standard error of the mean.

Considering that Nck1’s Y105 was required for the Nck1-Lck interaction and inhibiting this interaction led to reduced CD3ε phosphorylation, we hypothesized that Y105 was required for the recruitment of Lck to CD3ε, thereby allowing CD3 phosphorylation. To test this idea, CD3ε was immunoprecipitated and analyzed for the presence of co-purified Lck. Upon TCR triggering, recruitment of Lck to CD3ε was observed in WT-Nck1-expressing cells but was strongly decreased in the Y105F-Nck1 cells (Figure 6C). It is important to note that in resting T cells, Lck migrates at approximately 56 kDa, and upon phosphorylation at serine 59, it shifts to a ∼60 kDa activated form. This phosphorylation is mediated by phospholipase C and the MAP kinase ERK1/2 following TCR engagement.³⁰ Accordingly, Lck activation typically appears as two adjacent bands (56 and 60 kDa) on immunoblots (Figure 6A). Interestingly, co-immunoprecipitation of Lck with Nck1 and CD3ε reveals a single protein band of identical molecular weight in both resting and TCR-stimulated T cells. This suggests that the pool of Lck associated with Nck1 and CD3ε may not be phosphorylated by ERK1/2. Overall, Y105 on Nck1 is required for the interaction of Nck1 with Lck and for the stable interaction of Lck with TCR. Accordingly, it was confirmed that Nck1 helps Lck bind to the TCR.^8,29

Lck is required for Nck1 phosphorylation

Y105 of Nck1 is phosphorylated (Figure 2), and a phenylalanine mutation has drastic consequences for TCR signaling. Thus, the ability of Lck to phosphorylate Y105 of Nck1 was tested. To this end, an in vitro Lck kinase assay was performed using WT-Nck1. Nck1 was purified using an anti-Nck1 antibody from WT Jurkat cells and as a control from N1KO cells. The kinase assay was performed by adding recombinant Lck and ATP. Nck1 was only phosphorylated in the presence of both, Lck and ATP, but not in N1KO cells (Figure S3). As expected, Nck1 was phosphorylated in wild type Jurkat T cells but not in Lck-deficient Jurkat cells after TCR stimulation (Figure 7A). To further confirm that Lck is required for Nck1 phosphorylation, an Lck-specific inhibitor was applied during TCR stimulation, while a ZAP70 inhibitor was used as a control. Jurkat T cells were stimulated with an anti-TCR antibody (C305) for 10 min in the presence of either the Lck inhibitor or the ZAP70 inhibitor. As expected, treatment with the Lck inhibitor efficiently abrogated Nck1 phosphorylation (Figure 7B). In contrast, Nck1 phosphorylation was not impaired by treatment with the ZAP70 inhibitor. Collectively, these findings show that Y105 of Nck1 is phosphorylated by Lck upon TCR activation.

Figure 7.

The requirement of Lck for Nck1 phosphorylation

(A) Parental Jurkat and Lck-deficient (Jcam1.6) cells were stimulated and subjected to Nck1 immunoprecipitation, followed by immunoblotting for phosphotyrosine (4G10) and Nck1. Representative blots and quantification of phospho-Nck1 normalized to total Nck1 are shown (n = 3).

(B) Jurkat T cells were stimulated with the anti-TCR antibody C305 for 10 min in the presence or absence of an Lck inhibitor (100 nM). In parallel, Jurkat cells were pretreated with a ZAP70 inhibitor (4 μM) and subsequently stimulated with the anti-TCR antibody C305 for 10 min. Cells were then lysed and immunoprecipitated with 1 μg of anti-Nck1 antibody, followed by immunoblotting with the indicated antibodies. Data are mean ± SEM (n = 3); statistical significance assessed by unpaired t test (∗p < 0.05). Abbreviations: Lck, lymphocyte-specific kinase; Lck in, Lck inhibitor; Nck, non-catalytic region of tyrosine kinase; SEM, standard error of the mean; ZAP70 in, ZAP70 inhibitor.

Discussion

In 2002, Nck was identified as a molecule recruited after TCR-induced conformational changes of CD3ε. The conformational change exposes the PRS of CD3ε, which is essential for initiating TCR-triggered downstream signaling.³ Since then, studies have focused on exploring the molecular mechanisms governing Nck recruitment to CD3ε upon TCR engagement. Ligand-binding to the TCR prior to CD3 phosphorylation exposes CD3ε′s PRS, enabling its interaction with the SH3.1 domain of Nck in a pull down assay using GST-SH3.1.³ The Nck-CD3ε interaction might be further stabilized by an interaction of the Nck’s SH2 domain with partially phosphorylated CD3ε, since co-IP of Nck with CD3ε requires partial CD3ε phosphorylation.¹⁵

Our study reveals the following key findings: (1) Nck1 is phosphorylated at low quantities after 5 min and peaked at 10 min after TCR activation; (2) Nck1 associates with CD3ε after 5 min of T cell stimulation followed by the disassociation of Nck1 from CD3ε when T cells are activated for 10 min; (3) mutation of Nck1’s Y105 inhibits disassociation of NCK1 from CD3 after 10 min of stimulation; (4) mutation of Y105 blocks the interaction between Nck1 and Lck, thereby disrupting the recruitment of Lck to CD3ε; and (5) Nck1 phosphorylation occurs in an Lck-dependent manner. Based on these findings, the molecular mechanisms by which the Y105F residue of Nck1 regulates TCR signaling were proposed. Upon TCR activation, Nck1 interacts with Lck. The Nck1-Lck complex is recruited to the TCR-CD3 complex, where the Y105 residue of Nck1 is phosphorylated by Lck. Phosphorylation of Y105 allows Lck to disassociate from CD3, and it may facilitate the binding of Lck to the ITAM motif of CD3, thereby initiating the phosphorylation of CD3 subunits.

Cryo-electron microscopy (cryo-EM) has shown ligand-induced conformational changes in the TCR when bound to tumor-derived pMHC complexes,³¹ unlike studies with artificially matured TCR bound to monomeric soluble pMHC, which reported no detectable changes.³² This discrepancy may stem from the ligand type, as monomeric pMHC may not induce significant rearrangements. Whether the TCR conformation seen by cryo-EM fully reflects its physiological state remains unclear.^26,33 Notably, reconstituting detergent-purified TCRs onto phospholipid nanodiscs alters TCR conformation, potentially promoting an active-like state.³⁴ Functional studies reveal only soluble di- or oligo-meric pMHCs, not monomeric forms, can trigger TCR activation.^35,36 Biochemical assays also show monomeric pMHC fails to induce conformational changes in the CD3ε PRS.^4,5 Thus, structures of TCR bound to monomeric pMHC likely do not represent functionally relevant conformations. Additionally, molecular dynamics and cell experiments demonstrate that resting TCR CDR loops lie over CD3 ectodomains, while activation reorients them upward.³⁷ Together, these findings support a dynamic model where di- or oligo-meric pMHC binding induces conformational changes in CD3 cytoplasmic tails, driving TCR activation. However, high-resolution structural studies examining TCR-pMHC complexes have failed to demonstrate substantial rigid-body conformational rearrangements of the TCR ectodomain following ligand binding, leading to the development of alternative mechanistic models that place greater emphasis on physical forces and membrane-level organization.³⁸ Rather than invoking a single global conformational switch, these models propose that TCR activation arises from processes such as receptor clustering, spatial exclusion of inhibitory phosphatases (including CD45; the kinetic segregation model), and/or engagement of CD4 or CD8 coreceptors that recruit Src-family kinases to the TCR-CD3 complex.³⁸ In this conceptual framework, TCR-pMHC engagement during T cell-APC interactions is thought to generate mechanical forces—such as tension or torque—that act on the TCR-CD3 complex, resulting in structural distortion and reorientation of CD3 cytoplasmic tails relative to the plasma membrane. These mechanically induced changes promote ITAM exposure and thereby initiate downstream signaling events.³⁹ This mechanism, commonly described as a receptor deformation or mechanosensing model, offers an explanation for how ligand binding can trigger functional rearrangements within CD3 cytoplasmic domains without necessitating major ectodomain structural alterations. Collectively, accumulating evidence propose an integrated model in which force-dependent cytoplasmic rearrangements, receptor clustering, membrane compartmentalization, and coreceptor-associated kinase activity act in concert to initiate TCR signaling, rather than relying on a single dominant conformational transition as the primary trigger.

In this study, Y105 located between the SH3.1 and SH3.2 domains of Nck1 was examined, to identify its potential role in mediating Nck1 interaction with CD3ε and its effects on downstream signaling. Evidence of Nck phosphorylation in Jurkat T cells has been reported.⁴⁰ In this study, immunoprecipitation using an anti-pTyr antibody confirmed that Nck1 was phosphorylated upon T cell activation. Three Tyr phosphorylation sites have been identified in Nck1; Y105, Y246, and Y268.¹⁸ We show that Y105 is evolutionarily conserved in vertebrates with the consensus sequence of E(D)RL(I)YDLNXPA, where x is any amino acid. Further, our MS analysis confirmed the phosphorylation of Y105 of Nck1 in Jurkat and in primary human CD4⁺ T cells.

In this study, phosphorylation of Nck1 was observed after 5 min of TCR stimulation and peaked at 10 min. The kinetics of Nck1 tyrosine phosphorylation suggests an important roel in regulating signal transduction. The Nck-TCR interaction increases within the first 5 min of TCR engagement, followed by dissociation after prolonged stimulation.^3,15,16,23 Consistent with previous reports, immunoprecipitation of the TCR and PLA revealed that the Nck1-CD3ε interaction peaked after 5 min of TCR stimulation and then decreased. This decrease was associated with elevated CD3ε and Nck1 phosphorylation. These results suggest that the level of CD3ε and/or Nck1 phosphorylation might regulate their association. Indeed, immunoprecipitation and PLA showed that the absence of Y105 phosphorylation in Nck1 inhibits Nck1 disassociation from CD3. These results suggest that the Y105 residue of Nck1 is a negative regulator of the Nck1-CD3 interaction.

The Y105F Nck1 mutation that inhibited tyrosine phosphorylation caused a strong decline in the phosphorylation of Nck1, CD3ε, CD3ς, ZAP70, and ERK1/2. This suggests that phosphorylation of Y105 is required for optimal TCR signaling. Likewise, TCR-induced CD69 expression in T cells was lower in Y105F-Nck1-expressing cells than in parental Jurkat- and WT-Nck1-expressing cells in response to TCR stimulation. The mutation at the Y105 residue of Nck1 inhibits CD69 expression to a similar degree as observed in Nck1-knockdown cells.¹⁴

The presence of Nck is required for efficient phosphorylation of CD3ε by Lck.²³ The interaction between Nck and Lck upon TCR triggering has been reported previously.^7,16,29,41 Lck and Nck are recruited to CD3ε upon TCR engagement.⁸ In addition to the PRS motif, which is required for binding to the Nck SH3.1 domain, CD3ε contains an ITAM motif and basic-rich sequence and an RK motif, all of them binding to Lck.⁸ Moreover, Nck is vital for efficient Lck recruitment to CD3ε. Possibly, Nck binds directly or indirectly to Lck to promote the recruitment of Lck to CD3ε, where Nck and Lck cooperate for optimal CD3 phosphorylation.^8,42,43 Interestingly, our study shows that Y105 mutation reduced the interaction between Nck1 and Lck and further inhibited the recruitment of Lck to CD3ε. Thus, the failure to phosphorylate various proteins in Y105F-Nck1 cells could be explained by the disruption of Lck recruitment to CD3ε. These results suggested that Y105 regulates the Nck1-Lck interaction, thereby regulating the recruitment of Lck to CD3ε. Although, direct examination of the Nck-Lck interaction was not conducted in this study, the Nck SH3.2 domain was previously identified as a binding site for the proline motif in the unique domain of Lck.⁴¹ In addition, the T cell-specific adaptor protein (TSAd) has been described as a bridge between Nck and Lck.²⁹ Interestingly, we show that Y105 phosphorylation of Nck1 is accomplished by Lck. In addition, the kinase c-Abl phosphorylates Nck1’s Y105 in human umbilical vein endothelial cells (HUVECs) in response to vascular endothelial growth factor.¹⁷ In addition, Nck is also phosphorylated by Fyn. Nck facilitates the recruitment of Fyn to VEGF receptor-2, where the activated Fyn phosphorylates Nck to initiate downstream signal transduction for actin reorganization in HUVEC cells.⁴⁴ Our study supports the notion that Nck is needed for efficient recruitment of Lck to the TCR for initiating TCR signaling.

In summary, this study reveals the molecular mechanism regulating Nck1 association with and dissociation from the TCR. This phenomenon relies on Lck-induced phosphorylation of the conserved Y105 residue present within the linker between the SH3.1 and SH3.2 domains of Nck1. Mutation of the Y105 residue of Nck1 impedes the dissociation of Nck1 from CD3ε and is accompanied by notable impairment of the activation of the downstream signaling proteins. Furthermore, Y105 of Nck1 is a critical phosphosite that mediates Nck1-Lck interactions. Thus, Y105 of Nck1 plays an important role in TCR signaling and activation.

Limitations of the study

Although we show that Nck1 Y105 is important for T cell activation, we did not elucidate how its phosphorylation alters the Nck1 structure or whether phospho-Y105 interacts with other proteins. In addition, the precise molecular mechanisms by which Nck1 Y105 contributes to T cell activation were not fully elucidated in this study. Furthermore, the proposed sequence of signaling events following TCR stimulation is likely more complex than depicted, involving coordinated contributions from receptor clustering, mechanical forces, and adaptor protein dynamics. Future biochemical and structural studies will be essential to obtain insight. Moreover, donor demographic information (including age and sex) was not available from buffy coat samples because samples were obtained in an anonymized manner. Thus, the potential influence of sex or other biological variables on the experimental outcomes could not be evaluated.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sutatip Pongcharoen (sutatipp@nu.ac.th).

Materials availability

The model generated in this study is available from the lead contact with a completed materials transfer agreement.

Data and code availability

• •The proteomics data have been deposited to the ProteomeXchange Consortium via the jPOST⁴⁵ partner repository with the dataset identifier, PXD080700 (Nck1 Y105 phosphorylation related to Figure 2G).
• •This study does not report original code.
• •Any additional information required to reanalyze the data reported in this study is available from the lead contact upon request.

Acknowledgments

This work was supported by the National Science Research and Innovation Fund (NSRF grant no. R2569B070 for S.P. and J.N.). S.P. has received research funding from the National Research Council of Thailand (NRCT) and Naresuan University (grant number N42A650331). Wolfgang Schamel was supported by the German Research Foundation (DFG) under Germany’s Excellence Strategy (EXC-2189, project ID: 390939984), the Excellence Initiative of the German Federal and State Governments (EXC-294), and in part by the Ministry for Science, Research, and Arts of the State of Baden-Württemberg and SFB1381 (project A9). We would like to thank Emma Christina Roche for English language revisions. We would like to thank Nattapon Simanon (Proteomics and Metabolomics Research Team, National Center for Genetic Engineering and Biotechnology, Thailand) for his assistance with the mass spectrometry analysis.

Author contributions

Conceptualization, J.N., P.P., W.W.S., and S.P.; methodology, P.W., P.P., A.N., P.S., and J.N.; investigation, P.W., P.P., A.N., P.S., J.N., and W.C.; writing – original draft, J.N. and P.W.; writing – review and editing, J.N., P.W., P.P., W.C., W.W.S., and S.P.; visualization, P.W. and J.N.; formal analysis, P.W., J.N., and W.C.; supervision, S.P., J.N., and W.W.S., project administration, S.P.; funding acquisition, S.P., J.N., and W.W.S.

Declaration of interests

The authors have no competing interests to declare.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used Perplexity AI, ChatGPT AI, and Editage in order to edit the language. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Graphical abstract was created in BioRender. ngoenkam, j. (2026) https://BioRender.com/u17l881.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-CD3 antibody (OKT3)	eBioscience	16-0037-81; RRID: AB_468854
Anti-CD3ε M20 antibody	Santa Cruz	sc-1127; RRID: AB_631128
Anti-GAPDH antibody	Cell Signaling	2118; RRID: AB_561053
Anti-idiotypic antibodies against Jurkat TCR (C305)	Merck	05–919; RRID: AB_417390
Anti-Lck antibody	Cell Signaling	2752; RRID: AB_2234649
Anti-Nck1 antibody	Cell Signaling	2319; RRID: AB_659872
Anti-Nck2 antibody	Abnova	H00008440-K
Anti-phospho-ERK1/2 antibody	Cell Signaling	9101; RRID: AB_331646
Anti-phospho-SLP-76(Y128) antibody	Merck	SAB4504654
Anti-phospho-ZAP70 (Y319) antibody	Cell Signaling	2701; RRID: AB_331600
Anti-phosphotyrosine (4G10)	Millipore	05–321; RRID: AB_309678
Anti-SLP76 antibody	Cell Signaling	4958; RRID: AB_2136713
Anti-ZAP70 antibody	Cell Signaling	2705; RRID: AB_2273231
APC anti–human IL-2 antibody	eBioscience	17-7029-82; RRID: AB_469492
PE anti-human CD69 antibody	BioLegend	310906; RRID: AB_314841
PerCP anti-human CD3 antibody	BioLegend	300428; RRID: AB_893298
Rabbit antiserum raised against CD3ζ 448	In-house	San Jose et al.46
Rabbit antiserum raised against phospho-CD3ε (phospho-εY1)	In-house	Dopfer et al.47
Chemicals, peptides, and recombinant proteins
4′,6-diamidino-2-phenylindole (DAPI)	Thermo Scientific™	D1306
Acetonitrile (ACN)	Thermo Scientific™	13474279
Adenosine triphosphate (ATP)	HiMedia	1HMD-RM439-25G
Ammonium bicarbonate (NH4HCO3)	Sigma-Aldrich	09830
Brefeldin A	BioLegend	420601
Brij 96	Sigma-Aldrich	SIALP6136-100G
Bromophenol blue	Thermo Scientific™	A18469.09–10G
Dithiothreitol	Thermo Scientific™	R0861
Ethylenediaminetetraacetic acid (EDTA)	Merck	60-00-4
Fetal calf serum (FBS)	Gibco	A5256701
Formic acid (FA)	Fisher Chemical™	CHM.F/1900/PB15
Glycerol	Sigma-Aldrich	G5516
Hydroxyethyl piperazineethanesulfonic acid (HEPES)	Gibco	15630080
Iodoacetamine	Merck	I1149
Kinase Buffer I	Abcam	ab189135
Lck inhibitor	Cayman Chemical	15135
Methanol	Merck	34860-1L-R
Paraformaldehyde	Sigma-Aldrich	158127
Penicillin/ Streptomycin	Gibco	15140122
Phalloidin-conjugated FITC	Abcam	ab235137
Phenylmethanesulfonyl fluoride (PMSF)	Sigma-Aldrich	P7626
Protease and Phosphatase Inhibitor Cocktail	Thermo Fisher	78440
Radioimmunoprecipitation assay (RIPA) buffer	Thermo Fisher	89900
Recombinant human Lck protein	SinoBiological	10043-H09B
Roswell Park Memorial Institute (RPMI) 1640 Medium	Gibco	11875093
Saponin	Sigma-Aldrich	47036
Sodium chloride (NaCl)	Sigma-Aldrich	7647-14-5
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	71725-50G
Sodium fluoride (NaF)	Merck	106449
Sodium orthovanadate (Na3VO4)	Merck	567540
Tris hydrochloride (Tris-HCl)	Roche	10812846001
Triton X-100	Sigma-Aldrich	X100-100 ML
Trypsin	Sigma-Aldrich	T4799-10G
ZAP70 inhibitor	Tocris Bioscience	ZAP 180013
Critical commercial assays
BD Cytofix/Cytoperm™	BD Biosciences	554714
Duolink® In Situ PLA Probe Anti-Mouse PLUS	Sigma-Aldrich	DUO92001-30RXN
Duolink® In Situ PLA Probe Anti-Rabbit MINUS	Sigma-Aldrich	DUO92005-30RXN
Sited-directed mutagenesis kit (QuickChange)	Agilent	R200523
Ingenio® Electroporation kit	Merck	MIR 50118
Experimental models: Cell lines
Jurkat, Clone E6-1	ATCC	TIB-152
J.Cam1.6 (derivative mutant of Jurkat)	ATCC	CRL-2063
Recombinant DNA
GeneArt® CRISPR Nuclease (OPF) vector	Invitrogen	A21174
Software and algorithms
BlobFinder program	Uppsala University	Version 3.2
ImageJ	Michael Mateas and Andrew Stern	Version 1.54
Prism software	GraphPad	Version 10
Mascot daemon	Matrix Science,	version 2.6.0
Deposited data
Mass spectrometry proteomics data	ProteomeXchange via jPOST	PXD080700
Other
Polyvinylidene fluoride (PVDF) membranes	Merck Millipore	T831.1
Protein-G Sepharose beads	GE Healthcare	17-0618-01
CD4+ T cell Isolation Kit	Miltenyi Biotec	130-096-533

Experimental model and study participant details

Cell lines

Jurkat T cells (human acute T-cell leukemia cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum and antibiotics under standard humidified conditions at 37°C with 5% CO₂. Cell line identity was authenticated by ATCC using short tandem repeat profiling before distribution. No additional authentication was performed after receipt. Mycoplasma testing was not performed during this study.

Nck1-deficient Jurkat cells (N1KO) and the reconstituted WT-Nck1 and Y105F-Nck1 Jurkat variants were generated from parental Jurkat cells as described in the method details section.

Human participants

Primary human CD4⁺ T cells were isolated from buffy coats obtained from two independent healthy adult donors provided by the Blood Bank Center, Naresuan University Hospital. Cells isolated from each donor were analyzed as independent biological replicates. No allocation to experimental groups was performed because the study used ex vivo primary cells for mechanistic analyses.

The study was approved by the Naresuan University Institutional Review Board (NU-IRB No. 573/59). Buffy coats were collected following institutional ethical guidelines and informed consent procedures implemented by the blood collection center. Donor demographic information (including age and sex) was not available because samples were obtained in an anonymized manner. Therefore, the potential influence of sex or other biological variables on the experimental outcomes could not be evaluated and is acknowledged as a limitation of this study.

Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation, and subsequently CD4⁺ T cells were purified as described in the method details section.

Method details

Antibodies and reagents

The following antibodies were used: rabbit antibodies specific for Nck1, Lck, actin, phospho-ZAP70 (Y319), phospho-SLP-76(Y128), phospho-ERK1/2, and glyceraldehyde-3-phosphate dehydrogenase from Cell Signalling Technology (Denver, MA, USA); mouse anti-idiotypic antibodies against Jurkat TCR (C305) and phosphotyrosine (4G10) (Millipore, Merk KGaA, Darmstadt, Germany); CD3 (OKT3) (eBioscience, San Diego, CA, USA); and goat anti-CD3ε M20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The rabbit antiserum raised against CD3ζ 448 and phospho-CD3ε (phospho-εY1) has been described previously.^41,42 Recombinant human Lck protein was purchased from SinoBiological (Sino Biological, Inc. Beijing, China), and the Kinase Buffer I was purchased from Abcam (Cambridge, UK; ab189135). The Lck-specific inhibitor and ZAP70-specific inhibitor were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Tocris Bioscience (Birstol, UK), respectively.

Generation of Nck1-deficient Jurkat T cells and Jurkat variant cells

Nck1-deficient Jurkat T cells (N1KO) were generated by transiently expressing Cas9, a single guide RNA (sgRNA) that targets the Nck1 region. Briefly, sgRNAs were cloned into the GeneArt® CRISPR Nuclease (OPF) vector (Invitrogen, #A21174) and electroporated into Jurkat T cells. Stable Nck1-deficient clones were obtained using a limiting dilution technique. Successful positive knockout individual clones were confirmed using fluorescence analysis and immunoblotting with an anti-Nck1 antibody. A clone with a complete deletion of endogenous Nck1 was selected and named N1KO. The oligonucleotide sequence of the sgRNA was as follows: Nck1 sgRNA sequence, 5’-GTCGTCAATAACCTAAATAC-3’. TCR expression levels in these clones were evaluated using flow cytometry.

To generate WT-Nck1 or Y105F-Nck1 cells, N1KO cells were reconstituted either with WT-Nck1 or Y105F-Nck1 expression plasmids via electroporation. The human Flag-tagged (C-terminal) WT-Nck1-encoding sequence contained in the pEBB plasmid was provided by Prof. Bruce J. Mayer (Connecticut University Health Centre, Farmington, CT, USA) and was used as a mutagenesis template. The mutant construct, Y105F-Nck1 (Y105F), was generated using a site-directed mutagenesis kit (QuickChange mutagenesis kit, Stratagene, La Jolla, CA, USA) with the following primers: 5’-GACCCAGGGGAACGT CTCTTTGACCTCAACATGCCCGC-3’ and 5’-GCGGGCATGTTGAGGTCAAAGAGACGTTCCCCTGG GTC-3’ for Y105F-Nck1. The mutation plasmid was confirmed using DNA sequencing with the following primers: 5’-TCAAGCCTCAGACAGTGGTTC-3’ and 5’-GCCACCTCTGCTTGTCCA-3’. N1KO cells were transfected with either a plasmid expressing WT-Nck1 or Y105F-Nck1 using an electroporator (Amaxa Nucleofector II and the manufacturer’s protocol X-005). All Jurkat variant cells were assessed for CD3 and Nck1 expression using flow cytometry and immunoblotting, respectively.

Human primary CD4⁺ T cell isolation

Primary human CD4⁺ T cells were then enriched from the PBMC fraction using a CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) based on magnetic-activated cell sorting (MACS), according to the manufacturer's protocol. The purity of the isolated CD4⁺ T-cell population was subsequently confirmed by flow cytometric analysis.

Cell culture, stimulation, immunoblotting, and immunoprecipitation

Parental Jurkat cells, N1KO cells, WT-Nck1 cells, Y105F-Nck1 cells, and primary human CD4⁺ T cells were cultured under the conditions described above. Before stimulation, cells were starved in 10 mM HEPES/RPMI 1640 medium without FBS for 1 h at 37°C. Subsequently, parental Jurkat cells and their variants were stimulated with the anti-TCR antibody C305 (1:50) for the indicated time points or left unstimulated. Primary human CD4⁺ T cells were stimulated with the anti-CD3ε antibody (OKT3) at 37°C for 10 min. The dilution of anti-TCR antibody (C305) at a ratio of 1:50 corresponds to ∼0.12 mg/ml C305, as previously described.¹⁵ Following stimulation, cells were lysed in 100 μl lysis buffer (20 mM Tris-HCl [pH 8], 137 mM NaCl, 2 mM EDTA, 10% glycerol, protease inhibitor, and phosphatase cocktails [Sigma-Aldrich, St. Louis, MO, USA], 1 mM PMSF, 5 mM iodoacetamine, 0.5 mM sodium orthovanadate, 1 mM NaF, and 0.3% Brij96V) for 30 min on ice and cell debris was eliminated by centrifugation at 13,000 rpm at 4°C for 15 min before harvesting the supernatant for further assays.⁴⁸

For immunoprecipitation, protein extracts were prepared as previously described¹⁵ and were either immunoprecipitated with 1 μg anti-CD3ϵ (OKT3) antibody or anti-Nck1 antibody or anti-phosphotyrosine (4G10) antibody simultaneously with protein-G Sepharose beads (Amersham Pharmacia Biotech, Little Chalfont, UK). The precipitated proteins were subjected to sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with the desired antibodies, followed by detection using a CCD camera (ImageQuant LAS 4000; GE Healthcare Life Sciences, Little Chalfont, UK). Protein band intensity was quantified using ImageJ software.

To inhibit the kinases Lck and ZAP70, cells were treated as previously described.⁴⁹ Briefly, Jurkat cells were starved in 10 mM HEPES/RPMI 1640 medium without FBS for 30 min at 37°C. Cells were then stimulated with the anti-TCR antibody C305 (1:50) for 10 min in the presence or absence of an Lck inhibitor (100 nM), or left untreated as a control. For ZAP70 inhibition, Jurkat cells were pretreated with or without a ZAP70 inhibitor (4 μM) for 30 min, followed by stimulation with the anti-TCR antibody C305 (1:50) for 10 min, or left unstimulated. Cells were subsequently lysed and immunoprecipitated with 1 μg of anti-Nck1 antibody, and further processed as described above.

Protein identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

After SDS-PAGE, the proteins of interest were excised from the gels and subjected to in-gel trypsin digestion. Protein gels were washed twice for 1 h each with 100 μl of 50% acetonitrile/25 mM ammonium bicarbonate (NH₄HCO₃) at 25°C. Gels were then incubated with 100 μl of 100% acetonitrile for 10 min followed by treating with reduction buffer (10 mM dithiothreitol in 25 mM NH₄HCO₃) at 25°C for 30 min. After incubation, alkylating buffer (10 mM iodoacetamide in 25 mM NH₄HCO₃) was added and incubated at 25°C in the dark for 30 min. Gels were washed with 100 μl of 100% acetonitrile for 10 min. Thereafter, gels were digested in 15 μl of 12.5 ng/μL trypsin (MS grade; Promega, USA) for 16 h at 37°C. The supernatant containing peptides was transferred to a new tube. Peptides were extracted twice for 15 min each in 25 μl of 0.1% formic acid. Finally, the extracted peptides were dried under a stream of nitrogen evaporator (Organisation, USA). Peptides were reconstituted with 15 μl of 0.1% formic acid before subjecting to LC-MS/MS analysis.

The LC-MS/MS system consisted of a liquid chromatography system (Dionex Ultimate 3000, RSLCnano System) combined with a captive spray ionization/mass spectrometer (Model Q-ToF Compact, Bruker, Germany) at the Proteomics Services, Faculty of Medical Technology, Mahidol University (Salaya Campus), Nakonpathum, Thailand. The liquid chromatography separation was conducted using a reversed-phase column (Acclaim PepMap RSLC 75 μm × 15 cm, nanoViper C18), protected by a guard column (C18 PepMap100, 300 μm × 5 mm, 5 μm), eluted at a flow rate of 0.3 μl/min under gradient conditions of 2–85% B over 50 min. Mobile phases A and B consisted of water/formic acid (99.9:0.1, v/v) and water/acetonitrile (20:80, v/v), respectively. Mass spectral data from 300 to 1500 m/z were collected in the positive ionisation mode. The most abundant peptide ions were subjected to tandem mass spectrometry (MS/MS) analysis to determine the peptide sequences.

Mascot daemon version 2.6.0 (Matrix Science, London, UK) was used to search for peptide sequences. Searching parameters included MS/MS ions followed by carbamidomethyl at cysteine residues as fixed modification, oxidation on methionine, phospho (ST), and phospho (Y) as variable modifications, peptide tolerance ±1.2 Da, and MS/MS fragment tolerance ±0.6 Da. Trypsin/phosphate was used to identify peptides with a maximum of two missed cleavages. The selected protein hits had a p-value of ≤0.05, and the proteins reported in this study had 95% confidence intervals.

In situ PLA assay

Cells were starved on diagnostic microscopic slides (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 h at 37°C in RPMI 1640 without FBS and left unstimulated or stimulated with the anti-TCR (C305) antibody at 37°C for 5 min. The cells were then fixed with 4% paraformaldehyde, permeabilised with 0.5% saponin, and blocked with a blocking solution. Subsequently, cells were co-incubated with goat anti-CD3ε (M20ϵ, Santa Cruz Biotechnology) and rabbit anti-Nck1 antibody (Cell Signalling Technology). A proximity ligation between the CD3ε and Nck1 molecules was performed according to the manufacturer’s instructions using the Duolink kit (Olink Bioscience, Uppsala, Sweden). Cell nuclei were stained with 4',6-diamidino-2-phenylindole, and a confocal microscope (C2; Nikon, Tokyo, Japan) was used for imaging and analysis. The number of PLA signal dots (red fluorescent signals) was scored using a BlobFinder (Uppsala University).

In vitro Lck kinase activity assay

For the Lck kinase assay, Nck proteins were immunoprecipitated from total cell extracts using an anti-Nck1 antibody coupled to protein-G Sepharose beads. The beads were washed three times with lysis buffer and finally with kinase assay buffer (40 mM HEPES [pH 7.4], 10 mM MgCl₂, and 3 mM MnCl₂). Twenty microlitres of kinase assay mix containing 1 μg of human Lck and 10 μM ATP in kinase assay buffer was added, and the solution was incubated at 30°C for 15 min. The kinase reaction was stopped by adding non-reducing electrophoresis sample buffer (6 ml 1 M Tris-HCl [pH 6.8], 50 ml 50% glycerol, 20 ml 10% SDS, 10 ml 1% bromophenol blue, 114 ml ddH₂0) and was analysed using 12% gel SDS-PAGE. Nck phosphorylation was detected using immunoblotting with an anti-pTyr (4G10) antibody.

Flow cytometry

The effects of the Y105F-Nck1 on TCR-induced expression of the activation marker CD69 were assessed. Cells (2.5 × 10⁵) were starved overnight in medium containing 1% serum at 37°C. After starvation, cells were stimulated with the anti-CD3ε Ab clone OKT3 (5 μg/ml) and anti-CD28 Ab (2 μg/ml) in RPMI without serum at 37°C and 5% CO₂ for 18 h. After incubation, the cells were stained with phycoerythrin-labelled anti-human CD69 antibody (BioLegend, San Diego, CA, USA) and with peridinin-chlorophyll-protein-labelled anti-human CD3 Ab (BioLegend). The stained cells were evaluated for the expression of CD69 using flow cytometry with gating on CD3-positive cells (FACSCalibur, Becton Dickinson, NJ, USA). Data were analysed using CellQuest Pro software, as previously described.⁵⁰

For intracellular staining of IL-2, cells (2.5 × 10⁵) were stimulated with the anti-CD3ε Ab clone OKT3 and anti-CD28 Ab in RPMI without serum at 37°C and 5% CO₂ for 18 h. For the last 3 h, Brefeldin A (BioLegend, San Diego, CA, USA) was added to the cultured cells until a final concentration of 10 μg/mL. Cells were harvested using centrifugation followed by fixation and permeabilisation using BD Cytofix/Cytoperm™ (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The cells were then incubated for 30 min with an allophycocyanin-conjugated anti-human IL-2 antibody (eBioscience, San Diego, CA, USA). After washing, the cells were analysed using a flow cytometer (FACSCalibur, Becton Biosciences, Franklin Lakes, NJ, USA).

Fluorescence microscopy

This study was conducted to assess the effect of the mutation of the Y105 residue of Nck1 on actin polymerisation, which is one of the key processes of T cell activation. F-actin staining and visualisation were performed as previously described.²³ Briefly, 2 × 10⁵ cells were seeded on chambered cover glasses (LabTek; Thermo Scientific) pre-coated with anti-CD3 antibody (1 μg/ml) and were then activated for 5 min at 37°C. After incubation, the cells were immediately fixed with 4% paraformaldehyde and permeabilised with 0·1% Triton X-100. After blocking with 10% foetal calf serum for 1 h, phalloidin-conjugated fluorescein isothiocyanate, FITC (Abcam, Cambridge, USA) (diluted in PBS at 1:200) was added to each chamber and further incubated for 20 min at room temperature. F-actin formation was observed under a fluorescence microscope (Nikon Eclipse Ti, Nikon®, Melville, NY) using NIS-Elements D software with 2560 × 1920 record pixels. The actin polymerisation was expressed as the corrected total fluorescence (CTCF) intensity, which was calculated using the following equation: CTCF = integrated density – (area of selected cell × mean fluorescence of background readings). The integrated density, area of the selected cells, and mean fluorescence of the background were quantified using ImageJ software.

Quantification and statistical analysis

Data are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments, unless otherwise indicated. Each experiment was performed in triplicate. Statistical analyses were performed using Student's t-test. A p-value < 0.05 was considered statistically significant.