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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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Confirmation of micro-FRET detection of AKAP79 binding to CaN and PKA by YFP acceptor photobleaching imaging of FRET CFP donor quenching in fixed COS7 cells. (A) Membrane colocalization of AKAP79–CFP (blue) and CaNA–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]), and relief of FRET CFP donor quenching by YFP acceptor photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔCaN (Δ315–360) (bottom). B) Membrane colocalization of AKAP79–CFP (blue) and PKA-RII–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]) and relief of FRET CFP donor quenching by YFP photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔPKA (1–361) (bottom). Bars, ∼15 μm.
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fig3: Confirmation of micro-FRET detection of AKAP79 binding to CaN and PKA by YFP acceptor photobleaching imaging of FRET CFP donor quenching in fixed COS7 cells. (A) Membrane colocalization of AKAP79–CFP (blue) and CaNA–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]), and relief of FRET CFP donor quenching by YFP acceptor photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔCaN (Δ315–360) (bottom). B) Membrane colocalization of AKAP79–CFP (blue) and PKA-RII–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]) and relief of FRET CFP donor quenching by YFP photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔPKA (1–361) (bottom). Bars, ∼15 μm.

Mentions: As in live cells, coexpression of AKAP79–CFP (Fig. 1 C, 1) with CaNA–YFP (Fig. 1 D) or PKA-RII–YFP (Fig. 1 E) resulted in membrane anchoring of CaN (Fig. 3 A) or RII (Fig. 3 B) to AKAP79, detected by the micro-FRET method in fixed cells. AKAP79–CaN and AKAP79–RII FRETC signals (pseudo-color) at specific membrane locations are shown in images gated to the presence of YFP acceptor (blue underlay) to represent relative FRET intensities on a scale of blue (no FRET) to green (low FRET) to red (high FRET) (Fig. 3, A and B, top, second panel from right). Sites of high relative FRETC intensity (orange-red) included vesicles and plasma membrane ruffles where the donor and acceptors were highly colocalized. Micro-FRET measurements of FRETNC in membrane structures for these fixed samples were very reproducible across multiple experiments (n = 8–12) for AKAP79–CFP paired with CaN–YFP (14.0 ± 1.2) or RII–YFP (13.0 ± 1.6). This FRETNC value for AKAP79–RII in fixed cells is very similar to that determined in live cells for RII (12.1 and 13.8; Table I), whereas the FRETNC value for AKAP79–CaNA in fixed cells is larger than that determined in live cells for CaN (1.4 and 2.8; Table I). Thus, quantitative comparison of FRET for the same protein complex in live cells versus fixed cells is difficult because fixation may trap the complex in a form with somewhat altered chromophore separation and orientations compared with in living cells. Nonetheless, we can measure FRETC in fixed cells for AKAP79 binding to PKA and CaN.


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)

Confirmation of micro-FRET detection of AKAP79 binding to CaN and PKA by YFP acceptor photobleaching imaging of FRET CFP donor quenching in fixed COS7 cells. (A) Membrane colocalization of AKAP79–CFP (blue) and CaNA–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]), and relief of FRET CFP donor quenching by YFP acceptor photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔCaN (Δ315–360) (bottom). B) Membrane colocalization of AKAP79–CFP (blue) and PKA-RII–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]) and relief of FRET CFP donor quenching by YFP photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔPKA (1–361) (bottom). Bars, ∼15 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172743&req=5

fig3: Confirmation of micro-FRET detection of AKAP79 binding to CaN and PKA by YFP acceptor photobleaching imaging of FRET CFP donor quenching in fixed COS7 cells. (A) Membrane colocalization of AKAP79–CFP (blue) and CaNA–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]), and relief of FRET CFP donor quenching by YFP acceptor photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔCaN (Δ315–360) (bottom). B) Membrane colocalization of AKAP79–CFP (blue) and PKA-RII–YFP (green) with micro-FRETC (pseudo-color/gated to YFP [blue underlay]) and relief of FRET CFP donor quenching by YFP photobleaching (ΔCFP pseudo-color/gated to CFPpost [blue underlay]) observed for AKAP79WT (top) but not ΔPKA (1–361) (bottom). Bars, ∼15 μm.
Mentions: As in live cells, coexpression of AKAP79–CFP (Fig. 1 C, 1) with CaNA–YFP (Fig. 1 D) or PKA-RII–YFP (Fig. 1 E) resulted in membrane anchoring of CaN (Fig. 3 A) or RII (Fig. 3 B) to AKAP79, detected by the micro-FRET method in fixed cells. AKAP79–CaN and AKAP79–RII FRETC signals (pseudo-color) at specific membrane locations are shown in images gated to the presence of YFP acceptor (blue underlay) to represent relative FRET intensities on a scale of blue (no FRET) to green (low FRET) to red (high FRET) (Fig. 3, A and B, top, second panel from right). Sites of high relative FRETC intensity (orange-red) included vesicles and plasma membrane ruffles where the donor and acceptors were highly colocalized. Micro-FRET measurements of FRETNC in membrane structures for these fixed samples were very reproducible across multiple experiments (n = 8–12) for AKAP79–CFP paired with CaN–YFP (14.0 ± 1.2) or RII–YFP (13.0 ± 1.6). This FRETNC value for AKAP79–RII in fixed cells is very similar to that determined in live cells for RII (12.1 and 13.8; Table I), whereas the FRETNC value for AKAP79–CaNA in fixed cells is larger than that determined in live cells for CaN (1.4 and 2.8; Table I). Thus, quantitative comparison of FRET for the same protein complex in live cells versus fixed cells is difficult because fixation may trap the complex in a form with somewhat altered chromophore separation and orientations compared with in living cells. Nonetheless, we can measure FRETC in fixed cells for AKAP79 binding to PKA and CaN.

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