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T cell receptor dwell times control the kinase activity of Zap70

Nature Immunology volume 16, pages 961–969 (2015)Cite this article

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Abstract

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

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Figure 1: Conformational changes in Zap70 kinase cause differences in HDX.
Figure 2: TCR binding, phosphorylation and mutation of I-B (Y315A-Y319A) induce the open conformation of Zap70.
Figure 3: Binding to any p-ITAM of the TCR-CD3 complex, phosphorylation and mutation of I-B (Y315A-Y319A) induce an open conformation and increased size of Zap70.
Figure 4: The conformation of Zap70 does not control its catalytic activity.
Figure 5: Y315–Y319 phenylalanine and alanine mutants bind p-ITAMs less and more efficiently than wild-type Zap70 kinase, respectively.
Figure 6: The closed conformation of Zap70 binds p-ITAMs more weakly than the open conformation does.
Figure 7: Y315-Y319 phenylalanine and alanine mutants have higher and reduced exchange rates with TCR MCs, respectively.

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Acknowledgements

We thank E. Komives, I. Masslenikov, C. Beuck and M. Harber for valuable discussions. We thank B.E. Jones (Eli Lilly) for access to the HDX-MS platform, J. Williamson (the Scripps Research Institute) for use of the BLI instrument and B. Varsanofieva for assistance with FRAP data acquisition. We thank S. Chalasani, M. Hetzer and T. Hunter for critical reading of the manuscript. Stable Jurkat cell lines were a gift from A. Weiss (University of California San Francisco, San Francisco, California, USA). HEK293T cells were a gift from M.M. Davis (Stanford University, Stanford, California). We thank the Nomis Foundation, the Waitt Foundation and the James B. Pendleton Charitable Trust for their support. This work was supported by the National Institutes of Health (1DP2GM105455-01) and the Salk Institute Cancer Center core facilities (CA014195) funded by the National Cancer Institute.

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Authors and Affiliations

  1. Nomis Center for Immunobiology and Microbial Pathogenesis & Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, California, USA

    Christian Klammt, Lucie Novotná, Dongyang T Li, Miriam Wolf, Amy Blount & Björn F Lillemeier

  2. Eli Lilly Inc., Lilly Biotechnology Center, San Diego, California, USA

    Kai Zhang & Jonathan R Fitchett

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Contributions

C.K. and B.F.L. designed the study and wrote the manuscript. C.K., K.Z. and J.R.F designed and carried out HDX-MS experiments and analyzed the results. B.F.L. carried out the kinase activity experiments. C.K. conducted biolayer interferometry and competition assays. L.N. and D.T.L. carried out FRAP analyses. C.K., L.N., M.W. and A.B. established and performed purification of recombinant proteins.

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Correspondence to Björn F Lillemeier.

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Integrated supplementary information

Supplementary Figure 1 Phosphorylation and p-ITAM binding have additive effects on Zap70 conformation.

Regions with changes in deuterium uptake in the phosphorylated (p) and receptor-bound state (cplx) are mapped on the auto-inhibited structure of Zap70. Increased (red) and reduced (blue) deuterium uptakes are color-coded (key). The resulting Zap70 conformations are indicated above each structure as closed (auto-inhibited) or open. The data represent three independent HDX-MS experiments per Zap70 form. Recombinant Zap70 mutants are abbreviated to YYDN (D461N), FFDN (D461N, Y315F, Y319F), AADN (D461N, Y315A, Y319A) and tSH2 (amino acids 1–256).

Source data

Supplementary Figure 2 TCR binding, phosphorylation and mutation of interdomain B (Y315A–Y319A) cause differences in hydrogen-deuterium exchange of Zap70.

Heat map of deuterium uptake changes for Zap70 and its mutants in the phosphorylated (p), receptor-bound (cplx) and phosphorylated and receptor bound (p and cplx) forms. Peptide regions with changes in uptake relative to YYDN are mapped onto the Zap70 amino acid sequence. Increased (red) and reduced (blue) deuterium uptakes are color-coded (key). Regions not covered by any peptides are labeled in gray. Amino acid numbers are shown as in the scale at the top. Protein domains are shown in color (second row with N-SH2, light green; I-A, yellow; C-SH2, green; I-B, purple; and KinD, beige). Recombinant Zap70 mutants are abbreviated to YYDN (D461N), FFDN (D461N, Y315F, Y319F), AADN (D461N, Y315A, Y319A) and tSH2 (amino acids 1–256).

Supplementary Figure 3 Zap70 structural models.

Models for the receptor-bound open (a) and phosphorylated open (b) conformations are based on observed deuterium uptake changes. The illustrations for the open conformations were generated using structures of the kinase domain (1U59) next to receptor-bound or non–receptor-bound tSH2 (2OQ1 and 1M61, respectively). Amino acids not covered by those structures are shown as an unstructured chain of dots. The N-terminal SH2 (N-SH2) is colored in light green, interdomain A (I-A) in yellow, C-terminal SH2 (C-SH2) in green, interdomain B (I-B) in purple and kinase domain (KinD) in beige. The p-ITAM is shown in orange.

Supplementary Figure 4 Recombinant nonphosphorylated Zap70 is purified and phosphorylated with recombinant Lck.

(a) 1 µg of each purified protein was analyzed by SDS-PAGE and stained with Coomassie. (b) Western blot analysis of recombinant Zap70 phosphorylation states monitoring p-Y292, p-Y319, p-Y492, p-Y493, total p-tyrosine and total protein. Zap70 YY and AA were expressed as SHP1 phosphatase domain fusion proteins to eliminate background phosphorylation. Kinase-dead (DN) Zap70 was used for structural and binding studies, and nonphosphorylated Zap70 with functional KinD was used for kinase assays. Recombinant Zap70 mutants are abbreviated to YY (wild-type), YYDN (D461N), FF (Y315F, Y319F), FFDN (D461N, Y315F, Y319F), AA (Y315A, Y319A), AADN (D461N, Y315A, Y319A) and tSH2 (amino acids 1–256). “Myc” indicates a C-terminal Myc tag, and “GST” indicates an N-terminal glutathione-S-transferase tag. CD, cytosolic domain; p and + indicate phosphorylation by Lck.

Supplementary Figure 5 The Y315–Y319 phenylalanine or alanine mutants have higher or reduced exchange rates with TCR microclusters in E6.1 Jurkat T cells.

(a) Zap70 microcluster formation in Jurkat P116 (left) and E6.1 (right) cells on anti-CD3ɛ (OKT3)-coated glass surfaces. Clusters were visualized by confocal microscopy at 100× magnification within 5 min of surface binding. (b) Jurkat E6.1 cells were transfected with Zap70-GFP fusion proteins. Jurkat E6.1 cells were activated on anti-CD3ɛ–coated glass surfaces, and FRAPConf measurements were performed within 5 min of surface binding. Zap70 forms are abbreviated to YY (wild type), FF (Y315F, Y319F), AA (Y315A, Y319A) and tSH2 (amino acids 1–256). FRAPConf data points (open symbols) and curves fitted to an exponential function. Error bars represent s.e.m. (with nYY = 7, nAA = 9, ntSH2 = 5 and nCD3ζ = 7). (c) Comparison of mobile fractions (top) and halftime recovery (bottom) for Zap70 and its mutants. N.A. indicates that analysis was not possible. Error bars for mobile fractions and t1/2 represent the 95% CI. Data shown here represent three independent experiments.

Source data

Supplementary Figure 6 Model of Zap70 TCR-binding dynamics and activation.

The schematic shows binding (continuous arrows) and conformation equilibria (dotted arrows) for wild-type Zap70 (YY; black), and the Y315F–Y319F (FF; blue) and Y315A–Y319A (AA; red) mutants. Arrow thickness correlates with the rates of forward and backward reactions. In brief, cytosolic Zap70 is in a dynamic equilibrium between the closed (autoinhibited; 1A) and open conformations (1B). p-ITAM binding results in either a bound-closed (2A) or a bound-open (2B) complex. The bound-closed complex is unstable, which commonly results in Zap70 release. Phosphorylation of Y315 and Y319 ‘stabilizes’ the open conformation and results in prolonged TCR association (3). Zap70 AA exists exclusively in an open conformation (1B) and always binds the TCR firmly (3). Once Zap70’s binding to the TCR is stabilized, it is fully phosphorylated and acquires catalytic activity.

Source data

Supplementary Figure 7 HDX-MS peptide coverage map for receptor-bound Zap70 with superimposed color-coded changes in deuterium uptake.

Peptides of p-CD3γCD–bound Zap70 (YYDNcplx) detected by HDX-MS are shown in relation to the Zap70 sequence. The protein domains are color-coded above the amino acid sequence. The N-terminal SH2 (N-SH2) is colored in light green, interdomain A (I-A) in yellow, C-terminal SH2 (C-SH2) in green, interdomain B (I-B) in purple and kinase domain (KinD) in beige. Increased (red) and reduced (blue) deuterium uptakes relative to that of YYDN alone are color-coded (key).

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Klammt, C., Novotná, L., Li, D. et al. T cell receptor dwell times control the kinase activity of Zap70. Nat Immunol 16, 961–969 (2015). https://doi.org/10.1038/ni.3231

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