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

Li H, Pink MD, Murphy JG, Stein A, Dell'Acqua ML, Hogan PG - Nat. Struct. Mol. Biol. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

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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) Kis 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 neurons21,29. (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 ratios21. 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.
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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) Kis 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 neurons21,29. (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 ratios21. 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.

Mentions: Knowledge of the CN–AKAP79 structure opens an opportunity to investigate the effect of systematically varying the affinity of this enzyme-scaffold interaction. Building on our earlier studies of the CN–PVIVIT structure and the rules that connect changes in the sequence of CN recognition sites in substrates to variations in affinity16, we designed a small panel of AKAP79 anchoring peptides whose affinities for CN span a wide range. The peptides contained the replacements I338P, T343A, or both. Wildtype and variant AKAP proteins are referred to by 7mer sequences to facilitate comparison with variant sequences of the variations introduced— for instance, a peptide containing the wildtype anchoring sequence is designated PIAIIIT, even though Pro337 is not part of the contact interface defined in the crystal structure. Competitive binding experiments in which these unlabeled AKAP variants, as GST fusion proteins, were used to displace labeled PVIVIT demonstrated that the relative affinities were PPAIIIT > PIAIIIT > PPAIIIA > PIAIIIA, covering more than a 100-fold range in Kd (Fig. 4a, 4b). Notably, the PPAIIIT variant, in which Ile338 was substituted with Pro, bound ~4-fold more strongly to CN than wildtype AKAP79.


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

Li H, Pink MD, Murphy JG, Stein A, Dell'Acqua ML, Hogan PG - Nat. Struct. Mol. Biol. (2012)

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) Kis 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 neurons21,29. (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 ratios21. 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.
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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) Kis 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 neurons21,29. (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 ratios21. 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.
Mentions: Knowledge of the CN–AKAP79 structure opens an opportunity to investigate the effect of systematically varying the affinity of this enzyme-scaffold interaction. Building on our earlier studies of the CN–PVIVIT structure and the rules that connect changes in the sequence of CN recognition sites in substrates to variations in affinity16, we designed a small panel of AKAP79 anchoring peptides whose affinities for CN span a wide range. The peptides contained the replacements I338P, T343A, or both. Wildtype and variant AKAP proteins are referred to by 7mer sequences to facilitate comparison with variant sequences of the variations introduced— for instance, a peptide containing the wildtype anchoring sequence is designated PIAIIIT, even though Pro337 is not part of the contact interface defined in the crystal structure. Competitive binding experiments in which these unlabeled AKAP variants, as GST fusion proteins, were used to displace labeled PVIVIT demonstrated that the relative affinities were PPAIIIT > PIAIIIT > PPAIIIA > PIAIIIA, covering more than a 100-fold range in Kd (Fig. 4a, 4b). Notably, the PPAIIIT variant, in which Ile338 was substituted with Pro, bound ~4-fold more strongly to CN than wildtype AKAP79.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

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