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Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy.

Oliveria SF, Gomez LL, Dell'Acqua ML - J. Cell Biol. (2002)

Bottom Line: The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins.However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available.Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

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Direct observation of a CaN–AKAP79–PKA ternary in living cells using CYFRET imaging. (A) Plasma membrane/cortical targeting of CaN and PKA mediated through binding to functionally separable sites on AKAP79. Colocalization of PKA-RII–YFP (green) and CaNA–myc with AKAP79WT–CFP (blue) is seen as white in the RGB composite (top). Selective loss of PKA-RII (green) but not CaNA–myc (red) from the plasma membrane by deletion of the AKAP79–RII binding site (ΔPKA), seen as pink in the RGB composite (middle). Selective loss of CaNA–myc (red) but not PKA-RII (green) from the plasma membrane by deletion of the AKAP79–CaNA binding site (ΔCaN), seen as blue-green in the RGB composite (bottom). (B) Plasma membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and untagged AKAP79WT (anti-AKAP79, TxRd, red), seen as white in RGB composite. (C) FRETC measured between CaNA–CFP and PKA-RII–YFP bound to AKAP79WT in a ternary complex at the plasma membrane (top). Loss of CaNA–CFP from the AKAP79-RII–YFP complex and loss of FRETC upon disruption of the CaN binding site (ΔCaN) (second row). Loss of RII–YFP from the AKAP79–CaNA–CFP complex and loss of FRETC upon disruption of the PKA binding site (ΔPKA) (third row). Membrane colocalization but no FRETC for CaNA–CFP and RII–YFP targeted through binding to separate AKAP molecules (ΔPKA + ΔCaN) (bottom). Bars, ∼20 μm.
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fig5: Direct observation of a CaN–AKAP79–PKA ternary in living cells using CYFRET imaging. (A) Plasma membrane/cortical targeting of CaN and PKA mediated through binding to functionally separable sites on AKAP79. Colocalization of PKA-RII–YFP (green) and CaNA–myc with AKAP79WT–CFP (blue) is seen as white in the RGB composite (top). Selective loss of PKA-RII (green) but not CaNA–myc (red) from the plasma membrane by deletion of the AKAP79–RII binding site (ΔPKA), seen as pink in the RGB composite (middle). Selective loss of CaNA–myc (red) but not PKA-RII (green) from the plasma membrane by deletion of the AKAP79–CaNA binding site (ΔCaN), seen as blue-green in the RGB composite (bottom). (B) Plasma membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and untagged AKAP79WT (anti-AKAP79, TxRd, red), seen as white in RGB composite. (C) FRETC measured between CaNA–CFP and PKA-RII–YFP bound to AKAP79WT in a ternary complex at the plasma membrane (top). Loss of CaNA–CFP from the AKAP79-RII–YFP complex and loss of FRETC upon disruption of the CaN binding site (ΔCaN) (second row). Loss of RII–YFP from the AKAP79–CaNA–CFP complex and loss of FRETC upon disruption of the PKA binding site (ΔPKA) (third row). Membrane colocalization but no FRETC for CaNA–CFP and RII–YFP targeted through binding to separate AKAP molecules (ΔPKA + ΔCaN) (bottom). Bars, ∼20 μm.

Mentions: The identified CaN (321–360) and PKA (388–409) binding sites on AKAP79 are in very close proximity to each other in primary sequence (Fig. 1 C, 1). Our FRET measurements above indicate that both proteins bound to AKAP79 are <50 Å from C/YFP at the AKAP COOH terminus. These findings raise the issue of whether PKA and CaN are bound to the same AKAP molecule simultaneously or if there is competition due to steric hindrance between the closely opposed binding sites. To address this issue, we sought to reconstitute CaN–AKAP79–PKA ternary complexes in COS7 cells. As a first step, we coexpressed AKAP79WT–CFP (Fig. 1 C, 1) and PKA-RII–YFP (Fig. 1 E) with myc-tagged CaNA (Dell'Acqua et al., 2002). Cells were fixed and labeled with anti-myc antibodies to visualize CaNA along with AKAP79–CFP and RII–YFP. These studies revealed that AKAP79WT–CFP, PKA-RII–YFP, and myc–CaNA were all colocalized in plasma membrane ruffles (Fig. 5 A, top). To show that this colocalization in plasma membrane structures was due to independent binding of CaN and PKA to their respective AKAP79 binding sites, we analyzed the ΔPKA (Fig. 1 C, 2) and ΔCaN (Fig. 1 C, 3) mutants. Deletion of the PKA anchoring site on AKAP79 led to cytoplasmic localization of RII–YFP but retention of AKAP79–CFP and myc–CaNA at the membrane (Fig. 5 A, middle). Deletion of the CaN anchoring site on AKAP79 had the opposite effect, causing cytoplasmic localization of myc–CaNA but maintaining membrane overlap for AKAP79–CFP and RII–YFP (Fig. 5 A, bottom). These results do not support a model of competition between PKA and CaN binding to AKAP79 and suggest that these proteins can bind independently to closely spaced, but functionally separate, binding sites on the AKAP.


Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy.

Oliveria SF, Gomez LL, Dell'Acqua ML - J. Cell Biol. (2002)

Direct observation of a CaN–AKAP79–PKA ternary in living cells using CYFRET imaging. (A) Plasma membrane/cortical targeting of CaN and PKA mediated through binding to functionally separable sites on AKAP79. Colocalization of PKA-RII–YFP (green) and CaNA–myc with AKAP79WT–CFP (blue) is seen as white in the RGB composite (top). Selective loss of PKA-RII (green) but not CaNA–myc (red) from the plasma membrane by deletion of the AKAP79–RII binding site (ΔPKA), seen as pink in the RGB composite (middle). Selective loss of CaNA–myc (red) but not PKA-RII (green) from the plasma membrane by deletion of the AKAP79–CaNA binding site (ΔCaN), seen as blue-green in the RGB composite (bottom). (B) Plasma membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and untagged AKAP79WT (anti-AKAP79, TxRd, red), seen as white in RGB composite. (C) FRETC measured between CaNA–CFP and PKA-RII–YFP bound to AKAP79WT in a ternary complex at the plasma membrane (top). Loss of CaNA–CFP from the AKAP79-RII–YFP complex and loss of FRETC upon disruption of the CaN binding site (ΔCaN) (second row). Loss of RII–YFP from the AKAP79–CaNA–CFP complex and loss of FRETC upon disruption of the PKA binding site (ΔPKA) (third row). Membrane colocalization but no FRETC for CaNA–CFP and RII–YFP targeted through binding to separate AKAP molecules (ΔPKA + ΔCaN) (bottom). Bars, ∼20 μm.
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fig5: Direct observation of a CaN–AKAP79–PKA ternary in living cells using CYFRET imaging. (A) Plasma membrane/cortical targeting of CaN and PKA mediated through binding to functionally separable sites on AKAP79. Colocalization of PKA-RII–YFP (green) and CaNA–myc with AKAP79WT–CFP (blue) is seen as white in the RGB composite (top). Selective loss of PKA-RII (green) but not CaNA–myc (red) from the plasma membrane by deletion of the AKAP79–RII binding site (ΔPKA), seen as pink in the RGB composite (middle). Selective loss of CaNA–myc (red) but not PKA-RII (green) from the plasma membrane by deletion of the AKAP79–CaNA binding site (ΔCaN), seen as blue-green in the RGB composite (bottom). (B) Plasma membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and untagged AKAP79WT (anti-AKAP79, TxRd, red), seen as white in RGB composite. (C) FRETC measured between CaNA–CFP and PKA-RII–YFP bound to AKAP79WT in a ternary complex at the plasma membrane (top). Loss of CaNA–CFP from the AKAP79-RII–YFP complex and loss of FRETC upon disruption of the CaN binding site (ΔCaN) (second row). Loss of RII–YFP from the AKAP79–CaNA–CFP complex and loss of FRETC upon disruption of the PKA binding site (ΔPKA) (third row). Membrane colocalization but no FRETC for CaNA–CFP and RII–YFP targeted through binding to separate AKAP molecules (ΔPKA + ΔCaN) (bottom). Bars, ∼20 μm.
Mentions: The identified CaN (321–360) and PKA (388–409) binding sites on AKAP79 are in very close proximity to each other in primary sequence (Fig. 1 C, 1). Our FRET measurements above indicate that both proteins bound to AKAP79 are <50 Å from C/YFP at the AKAP COOH terminus. These findings raise the issue of whether PKA and CaN are bound to the same AKAP molecule simultaneously or if there is competition due to steric hindrance between the closely opposed binding sites. To address this issue, we sought to reconstitute CaN–AKAP79–PKA ternary complexes in COS7 cells. As a first step, we coexpressed AKAP79WT–CFP (Fig. 1 C, 1) and PKA-RII–YFP (Fig. 1 E) with myc-tagged CaNA (Dell'Acqua et al., 2002). Cells were fixed and labeled with anti-myc antibodies to visualize CaNA along with AKAP79–CFP and RII–YFP. These studies revealed that AKAP79WT–CFP, PKA-RII–YFP, and myc–CaNA were all colocalized in plasma membrane ruffles (Fig. 5 A, top). To show that this colocalization in plasma membrane structures was due to independent binding of CaN and PKA to their respective AKAP79 binding sites, we analyzed the ΔPKA (Fig. 1 C, 2) and ΔCaN (Fig. 1 C, 3) mutants. Deletion of the PKA anchoring site on AKAP79 led to cytoplasmic localization of RII–YFP but retention of AKAP79–CFP and myc–CaNA at the membrane (Fig. 5 A, middle). Deletion of the CaN anchoring site on AKAP79 had the opposite effect, causing cytoplasmic localization of myc–CaNA but maintaining membrane overlap for AKAP79–CFP and RII–YFP (Fig. 5 A, bottom). These results do not support a model of competition between PKA and CaN binding to AKAP79 and suggest that these proteins can bind independently to closely spaced, but functionally separate, binding sites on the AKAP.

Bottom Line: The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins.However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available.Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

ABSTRACT
Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A-kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

Show MeSH
Related in: MedlinePlus