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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 CaN binding to AKAP79 residues (321–360) in living cells using CYFRET microscopy. (A) Displacement of CaNA–YFP (green) from plasma membrane–targeted AKAP79 (anti-AKAP79, TxRd, red) by an untargeted AKAP79 CaN binding site peptide, AKAP79(321–360)–CFP (blue), seen in the RGB composite as red in membrane ruffles and CFP-blue/YFP-green overlap in the cytoplasm. (B) Colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79(321–360)–CFP (blue) in the cytoplasm of COS7 cells. (C) The AKAP79(321–360) CaN binding site confers CaNA binding activity on AKAP18 in living cells. Plasma membrane and Golgi targeting (CFP/YFP overlay) and direct binding (FRETC) of CaNA–YFP (green) to AKAP18(1–16)–AKAP79(321–360)–CFP (blue, bottom) but not AKAP18(1–16)–CFP (blue, top) in live COS7 cells. Bars, ∼20 μm.
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fig4: Direct observation of CaN binding to AKAP79 residues (321–360) in living cells using CYFRET microscopy. (A) Displacement of CaNA–YFP (green) from plasma membrane–targeted AKAP79 (anti-AKAP79, TxRd, red) by an untargeted AKAP79 CaN binding site peptide, AKAP79(321–360)–CFP (blue), seen in the RGB composite as red in membrane ruffles and CFP-blue/YFP-green overlap in the cytoplasm. (B) Colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79(321–360)–CFP (blue) in the cytoplasm of COS7 cells. (C) The AKAP79(321–360) CaN binding site confers CaNA binding activity on AKAP18 in living cells. Plasma membrane and Golgi targeting (CFP/YFP overlay) and direct binding (FRETC) of CaNA–YFP (green) to AKAP18(1–16)–AKAP79(321–360)–CFP (blue, bottom) but not AKAP18(1–16)–CFP (blue, top) in live COS7 cells. Bars, ∼20 μm.

Mentions: Our findings demonstrate the use of FRET to image binding between AKAP79 and CaN or PKA in both living and fixed cells, and from our controls with ΔCaN and ΔPKA mutants, we can infer that these interactions involve the appropriate AKAP binding sites. However, we wished to compliment these “loss of function” controls with a “gain of function” approach using the AKAP–CaN interaction as an example. Recently, we demonstrated that residues contained in 321–360 of AKAP79 can bind CaN in vitro and inhibit signaling in cells, suggesting that this region is sufficient for AKAP79–CaN anchoring (Dell'Acqua et al., 2002). To show that CYFRET observed between AKAP79 and CaN involves binding between AKAP79 residues 321–360 and the CaNA subunit, we constructed three additional CFP donors (AKAP79[321–360]–CFP, Fig. 1 C, 4; AKAP18[1–16]–CFP, Fig. 1 F, 1; and AKAP18[1–16]AKAP79[321–360]–CFP, Fig. 1 F, 2). In agreement with our previous studies, expression of the isolated CaN binding domain (321–360) fused to CFP (Fig. 1 C, 4) produced an untargeted protein found in both the cytoplasm and the nucleus (Fig. 4, A and B). This (321–360)–CFP construct bound to CaNA–YFP in the cytoplasm and prevented plasma membrane anchoring of CaNA–YFP to AKAP79WT (Texas red antibody staining) (Fig. 4 A). In parallel live cells, where untagged AKAP79 cannot be seen, colocalization of (321–360)–CFP and CaNA–YFP and FRETC were seen exclusively in the cytoplasm (Fig. 4 B). FRETNC measurements showed this FRET to be reproducible (13.3 ± 3.0; Table I).


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 CaN binding to AKAP79 residues (321–360) in living cells using CYFRET microscopy. (A) Displacement of CaNA–YFP (green) from plasma membrane–targeted AKAP79 (anti-AKAP79, TxRd, red) by an untargeted AKAP79 CaN binding site peptide, AKAP79(321–360)–CFP (blue), seen in the RGB composite as red in membrane ruffles and CFP-blue/YFP-green overlap in the cytoplasm. (B) Colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79(321–360)–CFP (blue) in the cytoplasm of COS7 cells. (C) The AKAP79(321–360) CaN binding site confers CaNA binding activity on AKAP18 in living cells. Plasma membrane and Golgi targeting (CFP/YFP overlay) and direct binding (FRETC) of CaNA–YFP (green) to AKAP18(1–16)–AKAP79(321–360)–CFP (blue, bottom) but not AKAP18(1–16)–CFP (blue, top) in live COS7 cells. Bars, ∼20 μm.
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Related In: Results  -  Collection

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fig4: Direct observation of CaN binding to AKAP79 residues (321–360) in living cells using CYFRET microscopy. (A) Displacement of CaNA–YFP (green) from plasma membrane–targeted AKAP79 (anti-AKAP79, TxRd, red) by an untargeted AKAP79 CaN binding site peptide, AKAP79(321–360)–CFP (blue), seen in the RGB composite as red in membrane ruffles and CFP-blue/YFP-green overlap in the cytoplasm. (B) Colocalization (CFP/YFP Overlay) and direct binding (FRETC) seen for CaNA–YFP (green) and AKAP79(321–360)–CFP (blue) in the cytoplasm of COS7 cells. (C) The AKAP79(321–360) CaN binding site confers CaNA binding activity on AKAP18 in living cells. Plasma membrane and Golgi targeting (CFP/YFP overlay) and direct binding (FRETC) of CaNA–YFP (green) to AKAP18(1–16)–AKAP79(321–360)–CFP (blue, bottom) but not AKAP18(1–16)–CFP (blue, top) in live COS7 cells. Bars, ∼20 μm.
Mentions: Our findings demonstrate the use of FRET to image binding between AKAP79 and CaN or PKA in both living and fixed cells, and from our controls with ΔCaN and ΔPKA mutants, we can infer that these interactions involve the appropriate AKAP binding sites. However, we wished to compliment these “loss of function” controls with a “gain of function” approach using the AKAP–CaN interaction as an example. Recently, we demonstrated that residues contained in 321–360 of AKAP79 can bind CaN in vitro and inhibit signaling in cells, suggesting that this region is sufficient for AKAP79–CaN anchoring (Dell'Acqua et al., 2002). To show that CYFRET observed between AKAP79 and CaN involves binding between AKAP79 residues 321–360 and the CaNA subunit, we constructed three additional CFP donors (AKAP79[321–360]–CFP, Fig. 1 C, 4; AKAP18[1–16]–CFP, Fig. 1 F, 1; and AKAP18[1–16]AKAP79[321–360]–CFP, Fig. 1 F, 2). In agreement with our previous studies, expression of the isolated CaN binding domain (321–360) fused to CFP (Fig. 1 C, 4) produced an untargeted protein found in both the cytoplasm and the nucleus (Fig. 4, A and B). This (321–360)–CFP construct bound to CaNA–YFP in the cytoplasm and prevented plasma membrane anchoring of CaNA–YFP to AKAP79WT (Texas red antibody staining) (Fig. 4 A). In parallel live cells, where untagged AKAP79 cannot be seen, colocalization of (321–360)–CFP and CaNA–YFP and FRETC were seen exclusively in the cytoplasm (Fig. 4 B). FRETNC measurements showed this FRET to be reproducible (13.3 ± 3.0; Table I).

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