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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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CFP/YFP micro-FRET microscopy imaging of AKAP79 binding to CaN and PKA in living cells. (A) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79–CFP WT but not ΔCaN (Δ315–360) (blue) in live COS7 cells. (B) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for PKA-RIIα–YFP (green) and AKAP79–CFP WT but not ΔPKA (1–361) (blue) in live COS7 cells. (C) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP79–YFP (green) that colocalize at the plasma membrane (CFP/YFP Overlay) but do not bind to each other. (D) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP18(1–16)–YFP (green) that colocalize (CFP/YFP Overlay) but do not bind to each other. Bars, ∼15 μm.
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fig2: CFP/YFP micro-FRET microscopy imaging of AKAP79 binding to CaN and PKA in living cells. (A) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79–CFP WT but not ΔCaN (Δ315–360) (blue) in live COS7 cells. (B) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for PKA-RIIα–YFP (green) and AKAP79–CFP WT but not ΔPKA (1–361) (blue) in live COS7 cells. (C) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP79–YFP (green) that colocalize at the plasma membrane (CFP/YFP Overlay) but do not bind to each other. (D) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP18(1–16)–YFP (green) that colocalize (CFP/YFP Overlay) but do not bind to each other. Bars, ∼15 μm.

Mentions: To facilitate FRET studies of the AKAP79 scaffold, we generated cDNA expression vectors for CFP and YFP COOH-terminal fusion proteins of AKAP79 (Fig. 1 C), CaNA catalytic subunit (Fig. 1 D), and PKA-RII regulatory subunit (Fig. 1 E) for expression in COS7 cells. COS7 cells do not express endogenous AKAP79 and have thus been used in transfection studies to reconstitute AKAP79, PKA, MAGUK, and glutamate receptor complexes, map the CaNA binding site on AKAP79, and study AKAP79 targeting (Colledge et al., 2000; Dell'Acqua et al., 2002; Gomez et al., 2002). Importantly, previous AKAP79 COS7 transfection studies are in good agreement with parallel experiments done in cultured neurons and brain extracts with endogenous proteins. Using micro-FRET subtraction to generate a corrected FRET (FRETC) image (see Materials and methods), we first set out to image binding between AKAP79 and CaNA. In agreement with earlier studies in fixed COS7 cells, expression of AKAP79WT–CFP (Fig. 1 C, 1) with CaNA–YFP (Fig. 1 D) resulted in targeting of CaN to plasma membrane ruffles and intracellular vesicles in living cells where extensive colocalization with the AKAP was seen (Fig. 2 A, CFP/YFP Overlay). In contrast, in cells coexpressing CaN–YFP with an AKAP79ΔCaN–CFP (Fig. 1 C, 3) mutant lacking the CaN binding site (Δ315–360) (Dell'Acqua et al., 2002), CaN was in the cytoplasm and not with membrane-targeted AKAP (Fig. 2 A). In cells expressing AKAP79WT–CFP and CaNA–YFP, FRETC (monochrome) was seen at membrane sites where the two proteins colocalized, but in cells expressing AKAP79ΔCaN, no FRET was detected.


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)

CFP/YFP micro-FRET microscopy imaging of AKAP79 binding to CaN and PKA in living cells. (A) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79–CFP WT but not ΔCaN (Δ315–360) (blue) in live COS7 cells. (B) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for PKA-RIIα–YFP (green) and AKAP79–CFP WT but not ΔPKA (1–361) (blue) in live COS7 cells. (C) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP79–YFP (green) that colocalize at the plasma membrane (CFP/YFP Overlay) but do not bind to each other. (D) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP18(1–16)–YFP (green) that colocalize (CFP/YFP Overlay) but do not bind to each other. Bars, ∼15 μm.
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fig2: CFP/YFP micro-FRET microscopy imaging of AKAP79 binding to CaN and PKA in living cells. (A) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79–CFP WT but not ΔCaN (Δ315–360) (blue) in live COS7 cells. (B) Plasma membrane/cortical colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for PKA-RIIα–YFP (green) and AKAP79–CFP WT but not ΔPKA (1–361) (blue) in live COS7 cells. (C) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP79–YFP (green) that colocalize at the plasma membrane (CFP/YFP Overlay) but do not bind to each other. (D) Negative control showing no FRETC for AKAP79–CFP (blue) and AKAP18(1–16)–YFP (green) that colocalize (CFP/YFP Overlay) but do not bind to each other. Bars, ∼15 μm.
Mentions: To facilitate FRET studies of the AKAP79 scaffold, we generated cDNA expression vectors for CFP and YFP COOH-terminal fusion proteins of AKAP79 (Fig. 1 C), CaNA catalytic subunit (Fig. 1 D), and PKA-RII regulatory subunit (Fig. 1 E) for expression in COS7 cells. COS7 cells do not express endogenous AKAP79 and have thus been used in transfection studies to reconstitute AKAP79, PKA, MAGUK, and glutamate receptor complexes, map the CaNA binding site on AKAP79, and study AKAP79 targeting (Colledge et al., 2000; Dell'Acqua et al., 2002; Gomez et al., 2002). Importantly, previous AKAP79 COS7 transfection studies are in good agreement with parallel experiments done in cultured neurons and brain extracts with endogenous proteins. Using micro-FRET subtraction to generate a corrected FRET (FRETC) image (see Materials and methods), we first set out to image binding between AKAP79 and CaNA. In agreement with earlier studies in fixed COS7 cells, expression of AKAP79WT–CFP (Fig. 1 C, 1) with CaNA–YFP (Fig. 1 D) resulted in targeting of CaN to plasma membrane ruffles and intracellular vesicles in living cells where extensive colocalization with the AKAP was seen (Fig. 2 A, CFP/YFP Overlay). In contrast, in cells coexpressing CaN–YFP with an AKAP79ΔCaN–CFP (Fig. 1 C, 3) mutant lacking the CaN binding site (Δ315–360) (Dell'Acqua et al., 2002), CaN was in the cytoplasm and not with membrane-targeted AKAP (Fig. 2 A). In cells expressing AKAP79WT–CFP and CaNA–YFP, FRETC (monochrome) was seen at membrane sites where the two proteins colocalized, but in cells expressing AKAP79ΔCaN, no FRET was detected.

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