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Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response - PubMed

  • ️Fri Jan 01 2016

Multisite Phosphorylation Modulates the T Cell Receptor ζ-Chain Potency but not the Switchlike Response

Himadri Mukhopadhyay et al. Biophys J. 2016.

Abstract

Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR ζ-chain is a homodimer subunit that contains six ITAMs (12 sites) and exhibits a number of properties that are predicted to be sufficient for a switchlike response. We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1
Figure 1

Cellular reconstitution of multisite phosphorylation of the T cell receptor ζ-chain. (A) Schematic of reconstituted signaling proteins. The substrate is a CD2-TCRζ-chain chimera that contains six phosphorylation sites distributed on three ITAMs (orange) that dimerizes as a result of a disulfide bond in the ζ-chain transmembrane domain (i.e., the substrate is a receptor dimer that contains 12 phosphorylation sites). The substrate is phosphorylated by the membrane-anchored kinase Lck and dephosphorylated by the transmembrane phosphatase CD148. The cytosolic kinase ZAP-70 can bind to phosphorylated ITAMs by SH2 domains. (B) Combinations of these components were transfected into the nonhematopoietic HEK293 cell line and molecular expression was detected with flow cytometry 24 h posttransfection. A k-means clustering algorithm classified the cells as either positive (red, 25%) or negative (blue, 75%) for transfected components (see Fig. S2 for two-dimensional projections). (C) Cells transfected with the indicated components were incubated with increasing concentrations of the tyrosine phosphatase inhibitor pervanadate for 30 min (x axis) before total phosphorylation of the ζ-chain was determined (shaded rectangle highlights the specificity range). (D) Phosphorylation time course for reconstitution of Lck, CD148, and ζ-chain indicates that steady-state phosphorylation is achieved by ∼15–20 min. See Materials and Methods for experimental details. To see this figure in color, go online.

Figure 2
Figure 2

Multisite phosphorylation of the T cell receptor ζ-chain enhances potency but not the switchlike response. (A) Phosphorylation profiles of reconstituted signaling modules containing Lck, CD148, and either wild-type ζ-chain containing all three ITAMs (ζ123) or all possible combination of ITAM mutations that replace the two ITAM tyrosines with phenylalanine. A Hill function is fit to all curves to produce estimates of the (B) the Hill number and (C) potency (EC50). (D) Expression of ζ-chain, Lck, CD148, and the ratio of Lck to CD148 at the single cell level is comparable for the seven reconstituted signaling modules (see Fig. S4 for comparison of mean expression and percent positive). Representative data (A and D) and averaged parameters (B and C) are normalized to the index module (Lck, CD148, and ζ123) with error bars indicating mean ± SE. See Materials and Methods for details on normalization and statistical analysis. To see this figure in color, go online.

Figure 3
Figure 3

ZAP-70 enhances potency but not the switchlike response of ζ-chain phosphorylation. (A–C) Phosphorylation profiles of reconstituted signaling modules containing Lck, CD148, ζ-chain, and either wild-type ZAP-70 or ZAP-70∗ that contains point mutations to abolish SH2 domain binding and tyrosine kinase function. (D–F) Phosphorylation profiles of reconstituted signaling modules containing Lck, CD148, ZAP-70, and either wild-type ζ-chain or all possible combinations of ITAM mutations. Component expression is comparable in all signaling modules (Figs. S6 and S7). Representative data (A and D) and averaged parameters (B, C, E, and F) are normalized to an index module (Lck, CD148, ζ123, and ZAP-70) with error bars indicating mean ± SE. See Materials and Methods for statistical analysis. To see this figure in color, go online.

Figure 4
Figure 4

A phosphorylation-dependent enhancement in enzymatic efficiencies is sufficient to explain experimental results. (A) Multisite phosphorylation model showing the transitions between the concentration of phosphorylated substrate on the indicated number of ITAMs that is mediated by the kinase (E) and the phosphatase (shown as F). The model includes a fold-enhancement in the on-rate (λ) that is proportional to the number of phosphorylated ITAMs with the maximum increase λmax ∼ λ6. (B) Phosphorylation profiles for a 6-ITAM (ζ), 4-ITAM (ζX), and a 2-ITAM (ζXX) substrate calculated using λ = 3 (λmax = 729) showing the potency and Hill numbers. See the Supporting Material for computational details. To see this figure in color, go online.

Figure 5
Figure 5

A phosphorylation-dependent enhancement in the on-rate can arise from a disorder-to-order transition. (A) A polymer model of the ζ-chain predicts that binding of the kinase (or phosphatase) is impeded by entropic disorder (left) and, if phosphorylation imposes local order, this impedance will be reduced when the substrate is phosphorylated with the maximal reduction occurring when the substrate is fully phosphorylated (right). Larger catalytic domains will incur a larger entropic penalty of binding. (B) The degree of disorder is determined by the number of segments whose length is known as the Kuhn length. (C) Heat map showing λmax for different segment numbers (y axis) and for different ratios of the catalytic domain radius to Kuhn length (x axis). (D) Comparison of maximal enhancement over the Kuhn length (or persistence length) when the enzymatic binding site is at the center of the ζ-chain (blue) or at the membrane-distal tyrosine at position 152 (red). A Kuhn length of ∼0.5 or ∼2 amino acids corresponds to λmax ∼ 700. Black line in (C) corresponds to parameter range in (D). See Materials and Methods for computational details. To see this figure in color, go online.

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