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Balanced interactions of calcineurin with AKAP79 regulate Ca2+–calcineurin–NFAT signaling

Nature Structural & Molecular Biology volume 19, pages 337–345 (2012)Cite this article

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Abstract

In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca2+ channels and couples Ca2+ influx to activation of calcineurin and of its substrate, the transcription factor NFAT. Here we show that an IAIIIT anchoring site in human AKAP79 binds the same surface of calcineurin as the PxIxIT recognition peptide of NFAT, albeit more strongly. A modest decrease in calcineurin-AKAP affinity due to an altered anchoring sequence is compatible with NFAT activation, whereas a further decrease impairs activation. Counterintuitively, increasing calcineurin-AKAP affinity increases recruitment of calcineurin to the scaffold but impairs NFAT activation; this is probably due to both slower release of active calcineurin from the scaffold and sequestration of active calcineurin by 'decoy' AKAP sites. We propose that calcineurin-AKAP79 scaffolding promotes NFAT signaling by balancing strong recruitment of calcineurin with its efficient release to communicate with NFAT.

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Figure 1: Crystal structure of calcineurin in complex with AKAP79 peptide.
Figure 2: The calcineurin–AKAP79 complex in solution.
Figure 3: Equilibrium and kinetic measurements of calcineurin binding to AKAP79.
Figure 4: A high-affinity AKAP79 variant, PPAIIIT, does not support NFAT nuclear translocation in hippocampal neurons.
Figure 5: The high-affinity AKAP79 variant PPAIIIT does not couple Ca2+ influx to NFAT-dependent transcription in hippocampal neurons.
Figure 6: The high-affinity AKAP79 PPAIIIT variant decreases the rate of calcineurin dissociation in vitro and reduces the mobility of calcineurin in dendritic spines.
Figure 7: Model explaining the effects of altered calcineurin-AKAP79 anchoring interactions.

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Acknowledgements

We are grateful to the Department of Neurobiology, Harvard Medical School, for use of its spectrofluorometer, and to S. Lehrer, Boston Biomedical Research Institute, for access to the stopped-flow instrument. Diffraction data were collected at the Advanced Photon Source on Northeastern Collaborative Access Team beamline 24-ID-C, which is supported by award RR15-301 from the National Center for Research Resources at the US National Institutes of Health. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract No. DE-AC02-06CH11357. We thank A. Sorkin, University of Pittsburgh, for providing the tandem YFP-CFP construct. The work was supported by US National Institutes of Health grants AI40127 (to A. Rao and P.G.H.), MH080291 (to M.L.D.) and AI090428 (to H.L.). M.D.P. was supported in part by T32NS007083 and by an American Heart Association Predoctoral Fellowship from the Pacific Mountain Affiliate. J.G.M. was supported in part by T32HD041697.

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  1. Huiming Li and Matthew D Pink: These authors contributed equally to this work.

Authors and Affiliations

  1. Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital, Boston, Massachusetts, USA

    Huiming Li & Patrick G Hogan

  2. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

    Huiming Li

  3. Department of Pharmacology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA

    Matthew D Pink, Jonathan G Murphy & Mark L Dell'Acqua

  4. Program in Neuroscience, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA

    Matthew D Pink, Jonathan G Murphy & Mark L Dell'Acqua

  5. Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA

    Alexander Stein

  6. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Alexander Stein

  7. La Jolla Institute for Allergy & Immunology, La Jolla, California, USA

    Patrick G Hogan

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Contributions

H.L. prepared the recombinant calcineurin and AKAP79 proteins, carried out the X-ray crystallography, developed the FRET assay for calcineurin-AKAP interaction and performed equilibrium binding measurements. H.L. and P.G.H. analyzed data from these experiments. H.L. and A.S. carried out SEC-MALS measurements. H.L. and P.G.H. performed and analyzed the stopped-flow kinetic experiments. M.D.P., J.G.M. and M.L.D. carried out and analyzed the FRET and YFP/CFP ratio measurements in MDCK cells. M.D.P. and M.L.D. carried out and analyzed the NFAT localization and FRAP experiments with hippocampal neurons. J.G.M. and M.L.D. carried out and analyzed the transcriptional reporter experiments with hippocampal neurons. P.G.H. and H.L. drafted the manuscript, with significant contributions from M.L.D., M.D.P. and J.G.M.

Corresponding authors

Correspondence to Mark L Dell'Acqua or Patrick G Hogan.

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Li, H., Pink, M., Murphy, J. et al. Balanced interactions of calcineurin with AKAP79 regulate Ca2+–calcineurin–NFAT signaling. Nat Struct Mol Biol 19, 337–345 (2012). https://doi.org/10.1038/nsmb.2238

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