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

Abstract

In hippocampal neurons, the scaffold protein AKAP79 recruits the phosphatase calcineurin to L-type Ca(2+) channels and couples Ca(2+) 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.

Figure 1

Crystal structure of calcineurin in complex with AKAP79 peptide. (a) Location of the PxIxIT-like CN anchoring site within human AKAP79 protein and sequence comparison to the PVIVIT peptide. The numbering of the residues in AKAP79 and in PVIVIT peptide is shown above and below the sequences, respectively. (b) Overall structure of CN in complex with AKAP79 peptide. The key residues comprising the core CN-binding motif are labeled with arrowheads. (c) A 2F_o–F_c map contoured at 1σ showing the electron density surrounding the isoleucine stretch, Ile340–Ile342, of the IAIIIT CN-anchoring motif of AKAP79. (d) Superposition of the CN–AKAP79 peptide structure onto the previously solved CN–PVIVIT structure. The core CN-binding residues of AKAP79 are labeled as in b. (e) The hydrogen bond between Thr343 of AKAP79 and Asn330 of CN A recapitulates a critical contact^, in the CN–PVIVIT structure. (f) Side-by-side comparison of the CN–AKAP79 structure (left) and CN–PVIVIT structure (right) viewed from the “proline pocket” defined in the CN–PVIVIT structure. CN is shown in surface representation with residues in direct contact with the peptides colored orange, and the peptides are displayed in stick representation. The figures were prepared using Chimera and Coot.

Figure 2

The CN–AKAP79 complex in solution. (a) SEC-MALS analysis of CN alone (red), CN–AKAP complex (orange), and BSA standard (blue). Inset, corresponding SDS-polyacrylamide gel. (b) Alternative 1:1 complexes based on the two CN molecules in the crystal asymmetric unit. The figures were prepared using Chimera. (c) Competition of the indicated GST-AKAP79(333–348) fusion proteins with fluorescent PVIVIT peptide for binding to CN. The estimated K_is are AIAIIIT, 3.7 µM; PIAIIIT, 3.9 µM; PAAIIIT, 47 µM. Total competitor concentration (not free concentration) is plotted. Data shown are representative of three experiments. (d) and (e) Images of living MDCK cells showing membrane colocalization (turquoise in composite panels) and corrected CFP donor to YFP acceptor FRET gated to the CFP channel (FRETc; pseudocolor, blue=no FRET to red=high FRET) for the indicated AKAP79-CFP proteins (blue) and (d) CNA-YFP (green) or (e) PKA-RII-YFP (green). Scale bar=5 µm. (f) CFP, YFP, and FRETc images as above for a linked CFP-YFP construct. (g) Measurements of apparent FRET efficiency, used to correct CFP fluorescence intensity for quenching. (h) Corrected YFP/CFP fluorescence intensity ratios for experiments in panels d–f. Statistical comparisons were by one-way ANOVA with a Bonferroni post-hoc test, ***p<0.001 and ^nsp>0.05 compared to the linked CFP-YFP standard (n=10–41 cells).

Figure 3

Equilibrium and kinetic measurements of CN binding to AKAP79. (a) CN binding to AKAP79(333–408) was monitored by stopped-flow FRET measurements. The observed time course of binding at 22°C is shown for four CN concentrations. (b) A plot of plateau FRET intensities against CN concentrations. (c) The association rate constant k_on estimated from the data in panel a based on a model of reversible binding to a single class of sites. (d) Dissociation of the CN–AKAP79(333–408) complex at 22°C. In this AKAP79(333–408) protein, the RIIα-anchoring site was replaced by the AKAP-IS sequence^,. Data shown are representative of two experiments.

Figure 4

A high-affinity AKAP79 variant, PPAIIIT, does not support NFAT nuclear translocation in hippocampal neurons. (a) Recombinant GST, GST-tagged wildtype AKAP79(333–408) (PIAIIIT, lane 2), and the variants PIAIIIA, PPAIIIA, and PPAIIIT (lanes 3–5) were analyzed by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue. (b) K_is estimated in a competitive binding assay are PPAIIIT, 0.08 µM; wildtype, 0.36 µM; PPAIIIA, 12 µM; and PIAIIIA, 39 µM. Total competitor concentration (not free concentration) is plotted. Data shown are representative of three experiments. (c) KCl stimulus protocol previously shown to activate L-type Ca²⁺ channel signaling through CN in hippocampal neurons^,. (d) Summed intensity projection images of neuronal cell bodies and proximal dendrites in nonstimulated (NS) cultures and in cultures fixed at the indicated times after KCl stimulation. Transfection with control RNAi plasmid (pSil), AKAP150 RNAi plasmid, and RNAi-resistant expression plasmids is indicated. The paired images show YFP or AKAP-YFP (white), DAPI-stained nuclei (blue), and endogenous NFAT (red). (e) Time course of NFAT nuclear import after KCl stimulation, from experiments as in panel d, quantified as nucleus-to-cytoplasm mean fluorescence intensity ratios. Each point represents n=12–25 neurons, and in each case the data have been normalized to the value for nonstimulated cultures (t = 0 min). Statistical comparisons were by one-way ANOVA with a Bonferroni post-hoc test, *p<0.05 and **p<0.01 compared to AKAP79WT rescue.

Figure 5

The high-affinity AKAP79 variant PPAIIIT does not couple Ca²⁺ influx to NFAT-dependent transcription in hippocampal neurons. (a) Diagram of the 3×NFAT-AP1-CFP-NLS transcriptional reporter construct used for single-cell imaging of NFAT-dependent transcription. (b) Modified KCl stimulation protocol used to assay L-type Ca²⁺ channel activation of NFAT-dependent reporter gene transcription in hippocampal neurons. (c) Summed intensity projection images of neuronal cell bodies and proximal dendrites in nonstimulated (NS) cultures and in cultures fixed at the indicated times after KCl stimulation for neurons transfected with the 3×NFAT-AP1-CFP-NLS reporter along with the indicated RNAi, YFP, and AKAP79-YFP constructs as in Figure 4b. YFP fluorescence is in white and nuclear-localized CFP fluorescence is in pseudocolor with a relative scale from blue (low intensity) to red (high intensity). (d) and (e) Quantification of CFP reporter gene expression (d) 6 hr or (e) 16 hr after KCl stimulation (+) from experiments as in panel c, normalized to the nonstimulated condition (−). *p<0.05, **p<0.01, and ***p<0.001 by Student’s t-test compared to the respective nonstimulated condition (n=11–50 neurons).

Figure 6

The high-affinity AKAP79 PPAIIIT variant decreases the rate of CN dissociation in vitro and reduces the mobility of CN in dendritic spines. (a) and (b) Dissociation of CN from (a) wildtype AKAP79(333–408) and (b) its I338P variant at 36 °C. (c) Time-lapse images of wildtype AKAP79-YFP fluorescence recovery in single dendritic spines of rat hippocampal neurons in culture at the indicated times after photobleaching. A prebleach image (t = −10 s) is shown for comparison. (d) YFP fluorescence recovery in neurons cotransfected with CN A-YFP (CN-YFP) and CFP-tagged wildtype AKAP79 (AKAP79WT) or CFP-tagged AKAP79 variants (PPAIIIT, PPAIIIA) as indicated. CFP fluorescence is not shown. (e) and (f) Percent YFP fluorescence recovery plotted against time for the FRAP experiments illustrated in panels c and d and for similar experiments with the other AKAP79 variants PIAIIIA and ΔPIX (deletion of PIAIIIT). In the experiment with CN-YFP alone, transfection with the RNAi plasmid was omitted. Maximal percent recovery (mobile fraction) values stated in the text were calculated from the curves in panels e and f by fitting a single exponential function. Statistical p values stated in the text were determined by one-way ANOVA with a Bonferonni post-hoc test (n=10–28 neurons).

Figure 7

Model explaining the effects of altered CN-AKAP79 anchoring interactions. (a) With wildtype AKAP79, most CN-anchoring sites at the L-type Ca²⁺ channel are occupied. Both inactive and active CN are continually released from the scaffold sites and replaced, so that any activated CN rapidly becomes available to interact with NFAT. Release of inactive CN is not depicted. (b) Low-affinity anchoring sites are partially occupied. CN release from the scaffold sites is efficient, but only occupied sites contribute active CN at any instant in time, and thus the rate of release of active CN is reduced compared to wildtype AKAP79. (c) High-affinity anchoring sites are fully occupied. The rate of release of CN is somewhat reduced and, at any given time, a larger fraction of the active CN is in complex with other AKAP79 scaffold sites than in the case of wildtype AKAP79.