Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins

Abstract

Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca2+-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-θ and PLC-γ1. T cells from Itch- and Cbl-b–deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function–associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.

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Figure 1: Decreased PLC-γ1 levels correlate with T cell anergy.
Figure 2: Decreased PLC-γ1 and impaired Ca2+ mobilization correlate with T cell anergy.
Figure 3: E3 ubiquitin ligases of the HECT type induce ubiquitination and degradation of PLC-γ1.
Figure 4: Upregulation of E3 ligases in T cells subjected to sustained Ca2+ signaling.
Figure 5: Ionomycin-anergized T cells show decreased stability of the immunological synapse.
Figure 6: Cblb- and Itch-deficient T cells are resistant to anergy induction.

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Acknowledgements

We thank members of the Rao and Dustin laboratories for discussions, and T. Starr for the preparation of ICAM-1 and I-Ek for planar bilayer experiments. We also thank A. Altman, H. Band, J. Brugge, C. Joazeiro, M. Katan for advice and reagents. Supported by National Institutes of Health grants RO1-AI48213, RO1-AI40127 and RO3-HD39685 (to A.R.), RO1-AI50280 and R21-AI48542 (to Y.-C.L.) and AI-43542; an Irene Diamond Foundation grant (to M.L.D.); EMBO (V.H.); and the Cancer Research Institute (S.-H.I. and S.F.).

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Correspondence to Anjana Rao.

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Heissmeyer, V., Macián, F., Im, SH. et al. Calcineurin imposes T cell unresponsiveness through targeted proteolysis of signaling proteins. Nat Immunol 5, 255–265 (2004). https://doi.org/10.1038/ni1047

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