Structural and functional evidence that Nck interaction with CD3epsilon regulates T-cell receptor activity

Abstract

Recruitment of signaling molecules to the cytoplasmic domains of the CD3 subunits of the T-cell receptor (TCR) is crucial for early T-cell activation. These transient associations either do or do not require tyrosine phosphorylation of CD3 immune tyrosine activation motifs (ITAMs). Here we show that the non-ITAM-requiring adaptor protein Nck forms a complex with an atypical PxxDY motif of the CD3epsilon tail, which encompasses Tyr166 within the ITAM and a TCR endocytosis signal. As suggested by the structure of the complex, we find that Nck binding inhibits phosphorylation of the CD3epsilon ITAM by Fyn and Lck kinases in vitro. Moreover, the CD3epsilon-Nck interaction downregulates TCR surface expression upon physiological stimulation in mouse primary lymph node cells. This indicates that Nck performs an important regulatory function in T lymphocytes by inhibiting ITAM phosphorylation and/or removing cell surface TCR via CD3epsilon interaction.

Figure. 1

Domain architecture and mapping of the Nck2 SH3.1 binding site on the cytoplasmic tail of CD3ε (A) Domain boundaries of human CD3ε. CD3ε is a single transmembrane protein with an IgG-like ectodomain (shown as a blue ellipse). The 55-residue cytoplasmic domain of human CD3ε contains a PRS (red box) and an ITAM (blue box). The two tyrosine-phosphorylation sites of the ITAM are indicated with “P”. (B) Sequence alignment of the cytoplasmic segments of CD3ε. The conserved residues are shown in white letters on red background. The locations of the PRS (residues 158−164), the ITAM (residues 166−180) and two phosphorylation sites in the ITAM are also indicated. ITAM is overlapping with the T cell endocytosis signal in CD3ε as indicated by . (C) ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled cytoplasmic segment of CD3ε without (left) and with (right) 4-fold excess of Nck2 SH3.1. The residues with significant chemical shift changes ((ΔδH² + (ΔδN / 5)²)^1/2 > 0.4 ppm) are labeled. (D) Plot of the normalized chemical shift changes of the CD3ε tail upon binding to Nck2 SH3.1. The sequence of human CD3ε is shown in the same representation as in (B).

Figure 2

Structure of the CD3ε/Nck2-SH3.1 complex. (A) Stereo view of an ensemble of 20 final NMR structures of the CD3ε/Nck2-SH3.1 complex. Blue, red, and grey lines show the backbone traces of Nck2 SH3.1 (residues 4−59), CD3ε(residues 158−166), and disordered regions (N-terminal and linker). (B) Ribbon representation of the CD3ε/Nck2-SH3.1 complex. The complex of Nck2 SH3.1 (blue) and CD3ε (red) are shown in two orientations. (C) Interface of the CD3ε/Nck2-SH3.1 interaction. The Nck2 SH3.1 surface is shown in white except for hydrophobic residues, which are colored in yellow. The backbone of CD3ε tail and the sidechains interacting with Nck Sh3.1 are depicted with red sticks. The hydrogen bond between the Val161 carbonyl oxygen and Asn163 amide proton is shown with a black dotted line.

Figure 3

Dissociation constants and relative affinities of CD3ε and Nck2 SH3.1 mutants. Dissociation constants (K_D) of the indicated constructs to WT Nck2 SH3.1 or CD3ε segment (143−183) were determined by fitting the chemical shift changes due increasing concentration of binding partner (see Supplemental Figure 1 for detailed explanations). The bottom row shows the CD3ε/Nck2-SH3.1 mutant combination. Each value presents the average ± standard error of at least two measurements. Relative binding affinity (%) compared to the wt CD3ε/Nck2-SH3.1 interaction was calculated from the average dissociation constants. The dashed line indicates 100% relative affinity.

Figure 4

Detailed view of Asp165-Tyr166 binding site. The left panel is an electrostatic surface representation where positive and negative potentials (calculated with the program MOLMOL in blue and red, respectively). The right panel shows the sidechain orientation of Nck2 SH3.1 at the site. CD3ε is depicted with red stick in both panels.

Figure 5

Nck SH3.1 down-regulates phosphorylation of CD3ε by Fyn (A) In vitro tyrosine phosphorylation of the CD3ε segment 143−183 by the Fyn kinase. The CD3ε segment was phosphorylated by recombinant Fyn kinase for 30 min at 37 °C in the presence of the indicated concentrations of Nck2 SH3.1 or 10 μM of the Src kinase inhibitor, PP₂. The samples were subjected to immunoblotting with 4G10 Ab as shown in the inserted gel picture. The phosphorylation ratio of each Nck2 SH3.1 concentration relative to the condition in the absence of the protein was estimated by using Kodak 1D. These results are the representative of 2 individual experiments. (B) In vitro tyrosine phosphorylation of CD3ε mutants. The Tyr phosphorylation of CD3ε mutants by Fyn kinase in the presence and the absence of the indicated concentration of Nck2 SH3.1 were visualized by 4G10 Ab. The concentration of Nck2 SH3.1 was set two-fold higher than the equilibrium dissociation constants. The ratios of the band intensities with/without Nck SH3.1 are 40%, 10% and 80% for WT, Y177F, and Y166F, respectively. These results are the representative of 3 individual experiments.

Figure 6

Nck overexpression downregulates TCR expression. (A) FACS analysis of TCR surface expression. The upper panel shows the density plot of GFP and TCR expression levels for CD8+ cells without stimulation. Nonretroviral gene-expressing cells (GFP-) and retroviral gene-expressing cells (GFP+) cells are gated as indicated in the panel. The middle panels show the overlaid TCR expression histograms for empty vector (thin boundary, shaded) or WT Nck (thick boundary, not shaded) transfected cells after 3hrs stimulation with VSV8-peptide (1 nM)-loaded APCs at 37 °C. Surface expression of TCR was monitored by PE-conjugated MR9.4 anti-TCR Vβ₅ antibody. In the bottom panels, the mean fluorescence intensity of each sample relative to non-stimulated cells was calculated to determine the relative TCR expression. (B) TCR surface expression of LN CD8+ T cells transfected with either empty vector (Vec), wt Nck (WT), or mutants of Nck (K36A or W38A). Cells are un-stimulated or stimulated with VSV8-loaded APCs for 3 hrs at 37 °C. FACS analysis was performed in the same way as (A). The indicated values in (A) and (B) are averaged over three separate experiments with error bars representing ± SD of the mean. Statistical significance was evaluated by two-tailed Student's T-test.

Figure 7

Downregulation of TCR expression by stimulation with different concentrations of the APC-VsV8 complex. TCR surface expression of LN CD8+ T cells transfected with either empty vector (Vec), wt Nck (WT), or mutants of Nck (K36A or W38A) were tested without stimulation and 3hrs after the activation with different concentration of VsV8-loaded APCs are shown. FACS analysis was done in the same way as in Figure 6. The indicated values are average of three separate experiments with error bars representing ± SD of the mean.