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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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Targeting of SAP97 to the CaN–AKAP79–PKA membrane cytoskeleton signaling complex. (A) AKAP79 regulates plasma membrane targeting of SAP97. SAP97–YFP (green) is primarily cytoplasmic when expressed alone (−AKAP79) but is targeted to the membrane ruffles in association with AKAP79–CFP (+AKAP79, blue) in live COS7 cells (CFP/YFP Overlay). (B) AKAP79 regulates targeting of SAP97 to the cortical membrane cytoskeleton through determinants that lie COOH terminal to the AKAP NH2-terminal targeting domain. SAP97–YFP (green) is targeted to the membrane ruffles enriched in F-actin (Texas red–phalloidin, red) in association with AKAP79–CFP (WT[1–427], top row, colocalization of SAP97, AKAP, and F-actin, seen as white in RGB composite panel). SAP97–YFP (green) is cytoplasmic when expressed with AKAP79(153)–CFP (blue) NH2-terminal domain, which targets to F-actin membrane ruffles (N[1–153], middle row, colocalization of (1–153) with F-actin, seen as pink in RGB composite panel) or an untargeted CFP–(150–427) AKAP79 fragment (C[150–427], bottom row). (C) AKAP79 regulates cortical colocalization of SAP97 with CaN in COS7 cells. CaNA–YFP (green) and myc–SAP97 (TxRd, red) are both cytoplasmic when expressed alone (−AKAP79) but are colocalized at the plasma membrane with each other and AKAP79–CFP (blue) in cells expressing all three proteins (+AKAP79), seen as white in the RGB composite. (D) AKAP79 coordinates targeting of CaN, PKA, and SAP97 to the same plasma membrane structures in COS7 cells. AKAP79 (anti-79, Cy5, monochrome) mediated membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and myc–SAP97 (TxRd, red), seen as white in the RGB composite panel. Diagrams showing structures of the (E) AKAP79–CFP WT(1–427), N(1–153), and C(150–427) and (F) SAP97–YFP fusion proteins used above. Relevant cellular targeting, protein binding domains, and other structural motifs are indicated for both AKAP and SAP97 (see Results and Discussion for more details). Bars: (A and B) ∼15 μm; (C and D) ∼20 μm.
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fig6: Targeting of SAP97 to the CaN–AKAP79–PKA membrane cytoskeleton signaling complex. (A) AKAP79 regulates plasma membrane targeting of SAP97. SAP97–YFP (green) is primarily cytoplasmic when expressed alone (−AKAP79) but is targeted to the membrane ruffles in association with AKAP79–CFP (+AKAP79, blue) in live COS7 cells (CFP/YFP Overlay). (B) AKAP79 regulates targeting of SAP97 to the cortical membrane cytoskeleton through determinants that lie COOH terminal to the AKAP NH2-terminal targeting domain. SAP97–YFP (green) is targeted to the membrane ruffles enriched in F-actin (Texas red–phalloidin, red) in association with AKAP79–CFP (WT[1–427], top row, colocalization of SAP97, AKAP, and F-actin, seen as white in RGB composite panel). SAP97–YFP (green) is cytoplasmic when expressed with AKAP79(153)–CFP (blue) NH2-terminal domain, which targets to F-actin membrane ruffles (N[1–153], middle row, colocalization of (1–153) with F-actin, seen as pink in RGB composite panel) or an untargeted CFP–(150–427) AKAP79 fragment (C[150–427], bottom row). (C) AKAP79 regulates cortical colocalization of SAP97 with CaN in COS7 cells. CaNA–YFP (green) and myc–SAP97 (TxRd, red) are both cytoplasmic when expressed alone (−AKAP79) but are colocalized at the plasma membrane with each other and AKAP79–CFP (blue) in cells expressing all three proteins (+AKAP79), seen as white in the RGB composite. (D) AKAP79 coordinates targeting of CaN, PKA, and SAP97 to the same plasma membrane structures in COS7 cells. AKAP79 (anti-79, Cy5, monochrome) mediated membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and myc–SAP97 (TxRd, red), seen as white in the RGB composite panel. Diagrams showing structures of the (E) AKAP79–CFP WT(1–427), N(1–153), and C(150–427) and (F) SAP97–YFP fusion proteins used above. Relevant cellular targeting, protein binding domains, and other structural motifs are indicated for both AKAP and SAP97 (see Results and Discussion for more details). Bars: (A and B) ∼15 μm; (C and D) ∼20 μm.

Mentions: AKAP79 can bind MAGUK scaffold proteins including SAP97 and PSD-95 (Colledge et al., 2000). In particular, binding of AKAP79 to SAP97 is believed to recruit AKAP, PKA, and CaN to GluR1 subunits for the regulation of AMPA receptor phosphorylation (Colledge et al., 2000; Dell'Acqua et al., 2002; Tavalin et al., 2002). Using our transfection system, we set out to characterize the interaction between AKAP79 and SAP97 in living cells. In agreement with previous studies (Tiffany et al., 2000), SAP97–YFP expressed alone was predominantly cytoplasmic (Fig. 6 A, top). However, expression of SAP97–YFP with AKAP79–CFP resulted in substantial SAP97 targeting to membrane ruffles in live cells (Fig. 6 A, bottom). In fixed cells, these sites of membrane colocalization of SAP97 with AKAP79 were found to be enriched in cortical F-actin stained with phalloidin (Fig. 6 B, top). We have previously shown that the highly-basic NH2 terminus of AKAP79 is necessary and sufficient for binding to phosphatidylinositol-4,5-bisphosphate and F-actin in vitro and targeting to dendritic spines in neurons and membrane ruffles in COS7 cells (Fig. 6 E; Dell'Acqua et al., 1998; Gomez et al., 2002). AKAP79 binds to the SAP97 MAGUK SH3 and GK domains (Fig. 6 F); however, the MAGUK binding site of AKAP79 is less defined but is thought to be located COOH terminal to the targeting domain (1–153) and NH2 terminal to the CaN anchoring site (315–360) (Fig. 6 E; Colledge et al., 2000). Thus, we expressed SAP97–YFP with AKAP79(1–153)–CFP as an additional negative control to support the need for AKAP79–MAGUK binding in SAP97 cortical targeting. As seen previously (Gomez et al., 2002), AKAP79(1–153)–CFP targeted to membrane ruffles enriched with F-actin (Fig. 6 B, middle); however, SAP97–YFP remained cytoplasmic with little or no colocalization with cortical actin or the AKAP fragment (Fig. 6 B, bottom). Coexpression of SAP97–YFP with a COOH-terminal fragment of AKAP79 ([150–427]–CFP) that is unable to localize to membranes or bind actin (Fig. 6 E; Gomez et al., 2002) resulted in no targeting of either protein to cortical actin (Fig. 6 B, bottom). This confirmed that binding of SAP97 to determinants found COOH terminal to the AKAP targeting domain must be required for SAP97 targeting to membrane ruffles with AKAP79 (Fig. 6 E).


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)

Targeting of SAP97 to the CaN–AKAP79–PKA membrane cytoskeleton signaling complex. (A) AKAP79 regulates plasma membrane targeting of SAP97. SAP97–YFP (green) is primarily cytoplasmic when expressed alone (−AKAP79) but is targeted to the membrane ruffles in association with AKAP79–CFP (+AKAP79, blue) in live COS7 cells (CFP/YFP Overlay). (B) AKAP79 regulates targeting of SAP97 to the cortical membrane cytoskeleton through determinants that lie COOH terminal to the AKAP NH2-terminal targeting domain. SAP97–YFP (green) is targeted to the membrane ruffles enriched in F-actin (Texas red–phalloidin, red) in association with AKAP79–CFP (WT[1–427], top row, colocalization of SAP97, AKAP, and F-actin, seen as white in RGB composite panel). SAP97–YFP (green) is cytoplasmic when expressed with AKAP79(153)–CFP (blue) NH2-terminal domain, which targets to F-actin membrane ruffles (N[1–153], middle row, colocalization of (1–153) with F-actin, seen as pink in RGB composite panel) or an untargeted CFP–(150–427) AKAP79 fragment (C[150–427], bottom row). (C) AKAP79 regulates cortical colocalization of SAP97 with CaN in COS7 cells. CaNA–YFP (green) and myc–SAP97 (TxRd, red) are both cytoplasmic when expressed alone (−AKAP79) but are colocalized at the plasma membrane with each other and AKAP79–CFP (blue) in cells expressing all three proteins (+AKAP79), seen as white in the RGB composite. (D) AKAP79 coordinates targeting of CaN, PKA, and SAP97 to the same plasma membrane structures in COS7 cells. AKAP79 (anti-79, Cy5, monochrome) mediated membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and myc–SAP97 (TxRd, red), seen as white in the RGB composite panel. Diagrams showing structures of the (E) AKAP79–CFP WT(1–427), N(1–153), and C(150–427) and (F) SAP97–YFP fusion proteins used above. Relevant cellular targeting, protein binding domains, and other structural motifs are indicated for both AKAP and SAP97 (see Results and Discussion for more details). Bars: (A and B) ∼15 μm; (C and D) ∼20 μm.
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fig6: Targeting of SAP97 to the CaN–AKAP79–PKA membrane cytoskeleton signaling complex. (A) AKAP79 regulates plasma membrane targeting of SAP97. SAP97–YFP (green) is primarily cytoplasmic when expressed alone (−AKAP79) but is targeted to the membrane ruffles in association with AKAP79–CFP (+AKAP79, blue) in live COS7 cells (CFP/YFP Overlay). (B) AKAP79 regulates targeting of SAP97 to the cortical membrane cytoskeleton through determinants that lie COOH terminal to the AKAP NH2-terminal targeting domain. SAP97–YFP (green) is targeted to the membrane ruffles enriched in F-actin (Texas red–phalloidin, red) in association with AKAP79–CFP (WT[1–427], top row, colocalization of SAP97, AKAP, and F-actin, seen as white in RGB composite panel). SAP97–YFP (green) is cytoplasmic when expressed with AKAP79(153)–CFP (blue) NH2-terminal domain, which targets to F-actin membrane ruffles (N[1–153], middle row, colocalization of (1–153) with F-actin, seen as pink in RGB composite panel) or an untargeted CFP–(150–427) AKAP79 fragment (C[150–427], bottom row). (C) AKAP79 regulates cortical colocalization of SAP97 with CaN in COS7 cells. CaNA–YFP (green) and myc–SAP97 (TxRd, red) are both cytoplasmic when expressed alone (−AKAP79) but are colocalized at the plasma membrane with each other and AKAP79–CFP (blue) in cells expressing all three proteins (+AKAP79), seen as white in the RGB composite. (D) AKAP79 coordinates targeting of CaN, PKA, and SAP97 to the same plasma membrane structures in COS7 cells. AKAP79 (anti-79, Cy5, monochrome) mediated membrane colocalization of CaNA–CFP (blue), PKA-RII–YFP (green), and myc–SAP97 (TxRd, red), seen as white in the RGB composite panel. Diagrams showing structures of the (E) AKAP79–CFP WT(1–427), N(1–153), and C(150–427) and (F) SAP97–YFP fusion proteins used above. Relevant cellular targeting, protein binding domains, and other structural motifs are indicated for both AKAP and SAP97 (see Results and Discussion for more details). Bars: (A and B) ∼15 μm; (C and D) ∼20 μm.
Mentions: AKAP79 can bind MAGUK scaffold proteins including SAP97 and PSD-95 (Colledge et al., 2000). In particular, binding of AKAP79 to SAP97 is believed to recruit AKAP, PKA, and CaN to GluR1 subunits for the regulation of AMPA receptor phosphorylation (Colledge et al., 2000; Dell'Acqua et al., 2002; Tavalin et al., 2002). Using our transfection system, we set out to characterize the interaction between AKAP79 and SAP97 in living cells. In agreement with previous studies (Tiffany et al., 2000), SAP97–YFP expressed alone was predominantly cytoplasmic (Fig. 6 A, top). However, expression of SAP97–YFP with AKAP79–CFP resulted in substantial SAP97 targeting to membrane ruffles in live cells (Fig. 6 A, bottom). In fixed cells, these sites of membrane colocalization of SAP97 with AKAP79 were found to be enriched in cortical F-actin stained with phalloidin (Fig. 6 B, top). We have previously shown that the highly-basic NH2 terminus of AKAP79 is necessary and sufficient for binding to phosphatidylinositol-4,5-bisphosphate and F-actin in vitro and targeting to dendritic spines in neurons and membrane ruffles in COS7 cells (Fig. 6 E; Dell'Acqua et al., 1998; Gomez et al., 2002). AKAP79 binds to the SAP97 MAGUK SH3 and GK domains (Fig. 6 F); however, the MAGUK binding site of AKAP79 is less defined but is thought to be located COOH terminal to the targeting domain (1–153) and NH2 terminal to the CaN anchoring site (315–360) (Fig. 6 E; Colledge et al., 2000). Thus, we expressed SAP97–YFP with AKAP79(1–153)–CFP as an additional negative control to support the need for AKAP79–MAGUK binding in SAP97 cortical targeting. As seen previously (Gomez et al., 2002), AKAP79(1–153)–CFP targeted to membrane ruffles enriched with F-actin (Fig. 6 B, middle); however, SAP97–YFP remained cytoplasmic with little or no colocalization with cortical actin or the AKAP fragment (Fig. 6 B, bottom). Coexpression of SAP97–YFP with a COOH-terminal fragment of AKAP79 ([150–427]–CFP) that is unable to localize to membranes or bind actin (Fig. 6 E; Gomez et al., 2002) resulted in no targeting of either protein to cortical actin (Fig. 6 B, bottom). This confirmed that binding of SAP97 to determinants found COOH terminal to the AKAP targeting domain must be required for SAP97 targeting to membrane ruffles with AKAP79 (Fig. 6 E).

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