Research ArticleImmunology

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway

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Science Signaling  19 May 2015:
Vol. 8, Issue 377, pp. ra49
DOI: 10.1126/scisignal.2005596
  • Fig. 1 Experimental design for SILAC experiments.

    Human Jurkat p116 cells expressing ZAP-70AS were incubated with light or heavy stable isotope–labeled arginine and lysine amino acids, physically differentiating the two proteomes by a shift in molecular weight. Cells were treated with DMSO (light-labeled cells) or HXJ-42 (ZAP-70AS + inhibitor, heavy-labeled cells) for 90 s before stimulation. Each cell population was then incubated with OKT3 and OKT4 antibodies against CD3 and CD4, respectively, for 30 s before being treated with cross-linking immunoglobulin G (IgG) for the indicated times. A total of five biological replicates were performed. Sample preparation, mass spectrometry experiments, and data analysis were performed as indicated.

  • Fig. 2 Individual phosphorylation site changes with inhibitor treatment.

    (A to C) Comparison of the relative abundances of the indicated phosphopeptides for (A) Itk, Vav, and ERK; (B) CD3 ITAMs; and (C) Lck, ZAP-70, Nck, and PYK2 after cells preincubated with DMSO (control) or the ZAP-70AS inhibitor were left unstimulated (zero time point) or were stimulated through the TCR for the indicated times. A total of five biological replicates were performed, and the calculated average ratio and SD were plotted for each time point. *P < 0.05.

  • Fig. 3 Targets of ZAP-70–dependent negative feedback regulation in TCR-proximal signaling.

    A model of the changes in the phosphorylation of TCR signaling components that occurred after inhibition of ZAP-70 catalytic activity (represented by the blue line) illustrated as quantitative SILAC ratio heatmaps beside individual proteins, corresponding to the changes in phosphorylation between the inhibitor-treated and control cells across the four time points of TCR stimulation. Heatmaps were calculated from the average of five independent biological replicate experiments. Green represents increased phosphorylation, whereas red represents decreased phosphorylation in ZAP-70–inhibited cells than in DMSO-treated controls. White dots in the heatmap indicate a statistically significant difference (Q value < 0.05) for that time point and phosphopeptide. Below each heatmap square is a color bar representing the coefficient of variation for that point. Orange represents a high degree of variation, whereas black represents a low degree of variation among the replicate analyses. PIP3, phosphatidylinositol 3,4,5-trisphosphate; SHC, SH2 domain–containing transforming protein; Grb2, growth factor receptor–bound protein 2; GADS, Grb2-related adaptor downstream of SHC; ADAP, adhesion and degranulation–promoting protein; IP3, inositol 1,4,5-trisphosphate; MEK1/2, mitogen-activated protein kinase kinase 1 and 2. Asterisk (*) denotes phosphorylation sites previously described in the literature.

  • Fig. 4 Computational model of proximal TCR signaling.

    (A) In ZAP-70 null cells, active Lck [Lck(A)] sequentially phosphorylates [whereas phosphatases (P) dephosphorylate] the N- and C-terminal tyrosines in individual ITAMs, generating singly phosphorylated and later doubly phosphorylated ITAMs. (B) In the ZAP-70 reconstituted cells or in ZAP-70AS cells, ZAP-70 weakly binds to the singly phosphorylated ITAMs and strongly binds to the doubly phosphorylated ITAMs. (C) Active Lck phosphorylates the SH2-linker tyrosine residues Y315 and Y319 of ZAP-70, generating open ZAP-70. The subsequent phosphorylation by Lck of Y493 generates active ZAP-70. Active and open forms of ZAP-70 are dephosphorylated by phosphatases (Pz). Active and open forms of ZAP-70 phosphorylate a negative regulatory site (Y192) in Lck. The phosphatases denoted by “PE” dephosphorylate Lck to return it to its active state. Active Lck further regulates ITAM phosphorylation through this negative feedback loop.

  • Fig. 5 Correlation between the experimental and calculated SILAC ratios.

    (A) Comparison of the experimental (left) and calculated (right) SILAC ratios of the N- and C-terminal tyrosines in each ITAM for the ZAP-70 null/ZAP-70 reconstituted system as a ratio heatmap. (B) Comparison of the experimental (left) and calculated (right) SILAC ratios for tyrosine phosphorylation of ζ-chain ITAMs, Lck, and ZAP-70 proteins of the ZAP-70AS + inhibitor/ZAP-70AS system as a ratio heatmap. The experimental ZAP-70 null/ZAP-70 reconstituted SILAC ratios (left panel) were taken from a previous study (34). The calculated SILAC ratios (right) were determined for the presence of various biological effects. The “ZAP-70 bind” model captures the results when ZAP-70–mediated negative feedback is absent. The “ZAP-70 NF” model shows the results when ZAP-70–medited negative feedback occurs. The “ITAM and Lck” model illustrates the results with the fast initial phosphorylation of the N-terminal tyrosine residues of ITAMs by Lck and the subsequent binding of the SH2 domain of Lck to the N- and C-terminal tyrosines. The “ITAMs” model captures the results with ordered phosphorylation of ζ-chain ITAM tyrosines (scenarios 1, 2, and 3). The “ITAM and Lck” and “ITAMs” models include the ZAP-70–mediated negative feedback. The calculated SILAC ratios for the tyrosine phosphorylation of the Lck and ZAP-70 proteins of the ZAP-70AS + inhibitor/ZAP-70AS system are shown only for the ITAMs model. In the heatmap, red indicates a decrease in phosphorylation, black indicates no change, and green represents an increase in phosphorylation when comparing ZAP-70 null cells to ZAP-70 reconstituted cells or ZAP-70AS inhibited cells versus DMSO-treated control cells. The experimental phosphorylation of Y153 in the ZAP-70 null/ZAP-70 reconstituted SILAC ratio and of Y142 and Y123 in the ZAP-70AS + inhibitor/ZAP-70AS SILAC ratio were not detected, which is indicated by missing heatmaps. Asterisk (*) denotes phosphorylation sites previously described in the literature.

  • Fig. 6 Computational models that describe the asymmetry in ITAM phosphorylation.

    (A) Active Lck sequentially phosphorylates [and the phosphatases (P) dephosphorylate] the N- and C-terminal tyrosines in each ITAM, generating singly phosphorylated and later doubly phosphorylated ITAMs. Next, Lck binds through its noncatalytic SH2 domain to the phosphorylated N- and C-terminal tyrosines of each ITAM. Once bound, Lck rapidly phosphorylates the neighboring C- and N-terminal tyrosines of each ITAM, generating doubly phosphorylated ITAMs. (B) Kinetic scheme of the order of tyrosine phosphorylation in ITAMs according to scenarios 1, 2, and 3. k1 and k2 are the rates of production of singly phosphorylated N- and C-terminal tyrosines from their unphosphorylated forms, respectively, whereas k3 and k4 are the rates of production of doubly phosphorylated N- and C-terminal tyrosines from their singly phosphorylated forms, respectively.

  • Table 1 SILAC heatmap representation of the temporal changes in the extent of tyrosine phosphorylation of proteins associated with the KEGG (Kyoto Encyclopedia of Genes and Genomes) TCR signaling pathway.

    Heatmaps were calculated from five independent biological replicate experiments. Green represents enhanced phosphorylation in response to ZAP-70 inhibition, whereas red represents a decrease in the phosphorylation of the indicated proteins relative to that in DMSO-treated control cells. White dots in the heatmap indicate statistically significant differences (Q value < 0.05) for those time points. Below each heatmap square is a color bar representing the percentage confidence value (CV) for the indicated times. PI3K, phosphatidylinositol 3-kinase; RXR-α, retinoid X receptor α; MAPK14, mitogen-activated protein kinase 14.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/377/ra49/DC1

    Experimental procedures.

    Computational modeling.

    Fig. S1. Reproducibility of proteomic quantitative data in the absence of TCR stimulation.

    Fig. S2. Reproducibility of proteomic quantitative data for all TCR stimulation time points.

    Fig. S3. Validation of inhibitor specificity.

    Fig. S4. Binding of the SH2 domain of Lck to monophosphorylated TCR ζ-chain ITAM peptides.

    Fig. S5. The number of rebinding events between enzymes and substrates upon variation of different kinetic parameters.

    Fig. S6. Description of the ZAP-70 allosteric model and the results of calculations with this model.

    Fig. S7. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAM and Lck” model.

    Fig. S8. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAMs” model.

    Fig. S9. Sensitivity analysis of the kinetic parameters used in calculations for the ZAP-70 allosteric model (part I).

    Fig. S10. Sensitivity analysis of the kinetic parameters used in calculations for the ZAP-70 allosteric model (part II).

    Table S1. Complete list of sequence and phosphorylation site assignments of all identified phosphopeptides with corresponding SIC peak areas and statistics, protein association numbers, gene ontology, and KEGG functional annotation.

    Table S2. Complete list of phosphopeptides detected from every replicate and time point of TCR stimulation.

    Table S3. Fold change between HXJ-42–treated and DMSO-treated ZAP-70AS cells and Q values for peptides listed in Table 1.

    Table S4. Binding parameters for the Lck SH2 domain and monophosphorylated ζ ITAM peptides determined by isothermal titration calorimetry at 25°C.

    Table S5. Concentrations of species used in calculations for all models (volume = 1 μm3).

    Table S6. Reactions and kinetic parameters used in calculations for the ZAP-70–mediated negative feedback model and models used to reproduce an asymmetry in ITAM phosphorylation.

    Table S7. Reactions and kinetic parameters used in calculations for the ZAP-70 allosteric function model.

    Table S8. Sensitivity analysis of the concentrations of signaling molecules used in calculations for the “ITAM and Lck” and “ITAMs” models.

    Table S9. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAM and Lck” model.

    Table S10. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAMs” model.

    Table S11. Sensitivity analysis of the concentrations of signaling molecules used in calculations for the ZAP-70 allosteric model.

    Table S12. Sensitivity analysis of the kinetic parameters used in calculations for ZAP-70 allosteric model.

    References (7994)

  • Supplementary Materials for:

    The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway

    Hanna Sjölin-Goodfellow, Maria P. Frushicheva, Qinqin Ji, Debra A Cheng, Theresa A. Kadlecek, Aaron J. Cantor, John Kuriyan, Arup K. Chakraborty,* Arthur R. Salomon,* Arthur Weiss*

    *Corresponding author. E-mail: as{at}brown.edu (A.R.S.); arupc{at}mit.edu (A.K.C.); aweiss{at}medicine.ucsf.edu (A.W.)

    This PDF file includes:

    • Experimental procedures.
    • Computational modeling.
    • Fig. S1. Reproducibility of proteomic quantitative data in the absence of TCR stimulation.
    • Fig. S2. Reproducibility of proteomic quantitative data for all TCR stimulation time points.
    • Fig. S3. Validation of inhibitor specificity.
    • Fig. S4. Binding of the SH2 domain of Lck to monophosphorylated TCR ζ-chain ITAM peptides.
    • Fig. S5. The number of rebinding events between enzymes and substrates upon variation of different kinetic parameters.
    • Fig. S6. Description of the ZAP-70 allosteric model and the results of calculations with this model.
    • Fig. S7. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAM and Lck” model.
    • Fig. S8. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAMs” model.
    • Fig. S9. Sensitivity analysis of the kinetic parameters used in calculations for the ZAP-70 allosteric model (part I).
    • Fig. S10. Sensitivity analysis of the kinetic parameters used in calculations for the ZAP-70 allosteric model (part II).
    • Legends for tables S1 and S2
    • Table S3. Fold change between HXJ-42–treated and DMSO-treated ZAP-70AS cells and Q values for peptides listed in Table 1.
    • Table S4. Binding parameters for the Lck SH2 domain and monophosphorylated ζ ITAM peptides determined by isothermal titration calorimetry at 25°C.
    • Table S5. Concentrations of species used in calculations for all models (volume = 1 μm3).
    • Table S6. Reactions and kinetic parameters used in calculations for the ZAP-70–mediated negative feedback model and models used to reproduce an asymmetry in ITAM phosphorylation.
    • Table S7. Reactions and kinetic parameters used in calculations for the ZAP-70 allosteric function model.
    • Table S8. Sensitivity analysis of the concentrations of signaling molecules used in calculations for the “ITAM and Lck” and “ITAMs” models.
    • Table S9. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAM and Lck” model.
    • Table S10. Sensitivity analysis of the kinetic parameters used in calculations for the “ITAMs” model.
    • Table S11. Sensitivity analysis of the concentrations of signaling molecules used in calculations for the ZAP-70 allosteric model.
    • Table S12. Sensitivity analysis of the kinetic parameters used in calculations for ZAP-70 allosteric model.
    • References (7994)

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.60 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Complete list of sequence and phosphorylation site assignments of all identified phosphopeptides with corresponding SIC peak areas and statistics, protein association numbers, gene ontology, and KEGG functional annotation.
    • Table S2 (Microsoft Excel format). Complete list of phosphopeptides detected from every replicate and time point of TCR stimulation.

    [Download Tables S1 and S2]


    Citation: H. Sjölin-Goodfellow, M. P. Frushicheva, Q. Ji, D. A. Cheng, T. A. Kadlecek, A. J. Cantor, J. Kuriyan, A. K. Chakraborty, A. R. Salomon, A. Weiss, The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway. Sci. Signal. 8, ra49 (2015).

    © 2015 American Association for the Advancement of Science

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