Limits...
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.

Show MeSH

Related in: MedlinePlus

CFP/YFP FRET microscopy system for studying AKAP79 protein–protein interactions in living cells. (A) Model showing no FRET between A–CFP and B–YFP tagged proteins in the absence of binding. (B) CFP to YFP FRET (CYFRET) resulting in increased YFP acceptor emission and quenching of CFP donor emission seen when A–CFP and B–YFP tagged proteins are bound within 50 Å of each other. (C) CFP- and YFP-tagged (1) AKAP79 WT, (2) ΔPKA, (3) ΔCaN, and (4) (321–360) constructs used for CYFRET imaging of AKAP79 binding to CFP- and YFP-tagged (D) CaNAα and (E) PKA-RIIα. (F) (1) AKAP18(1–16) and (2) AKAP18(1–16)–AKAP79(321–360) hybrid CFP and YFP fusion proteins for CYFRET imaging of CaNA binding. AKAP79 anchors the CaNA,B holoenzyme through binding the catalytic CaNA subunit (Coghlan et al., 1995; Kashishian et al., 1998) and anchors the PKA heterotetrameric R2C2 holoenzyme through binding a surface created by dimerization of the of NH2 terminus of the regulatory subunit (RIIα/β) (Newlon et al., 2001). The NH2 and COOH termini of each protein are indicated by N and C or numbers starting with 1 at the NH2 terminus. The AKAP79 CaN (315–360, pink) and PKA (388–413, red) binding sites are indicated as are the AKAP79 (1–153, A, B, and C poly-basic, blue) and AKAP18 (1–16, NH2-terminal Gly-myristoylated, dual Cys-palmitoylated) membrane targeting domains.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172743&req=5

fig1: CFP/YFP FRET microscopy system for studying AKAP79 protein–protein interactions in living cells. (A) Model showing no FRET between A–CFP and B–YFP tagged proteins in the absence of binding. (B) CFP to YFP FRET (CYFRET) resulting in increased YFP acceptor emission and quenching of CFP donor emission seen when A–CFP and B–YFP tagged proteins are bound within 50 Å of each other. (C) CFP- and YFP-tagged (1) AKAP79 WT, (2) ΔPKA, (3) ΔCaN, and (4) (321–360) constructs used for CYFRET imaging of AKAP79 binding to CFP- and YFP-tagged (D) CaNAα and (E) PKA-RIIα. (F) (1) AKAP18(1–16) and (2) AKAP18(1–16)–AKAP79(321–360) hybrid CFP and YFP fusion proteins for CYFRET imaging of CaNA binding. AKAP79 anchors the CaNA,B holoenzyme through binding the catalytic CaNA subunit (Coghlan et al., 1995; Kashishian et al., 1998) and anchors the PKA heterotetrameric R2C2 holoenzyme through binding a surface created by dimerization of the of NH2 terminus of the regulatory subunit (RIIα/β) (Newlon et al., 2001). The NH2 and COOH termini of each protein are indicated by N and C or numbers starting with 1 at the NH2 terminus. The AKAP79 CaN (315–360, pink) and PKA (388–413, red) binding sites are indicated as are the AKAP79 (1–153, A, B, and C poly-basic, blue) and AKAP18 (1–16, NH2-terminal Gly-myristoylated, dual Cys-palmitoylated) membrane targeting domains.

Mentions: Using GFP as an expression tag to study protein targeting has become an indispensable tool of modern cell biology (Tsien, 1998). CFP and YFP variants of GFP have been developed that can be imaged separately in the same cell. In addition, energy can be transferred from an excited CFP donor to YFP acceptor through a nonradiative dipole–dipole interaction called FRET. There is a strong inverse relationship between FRET and chromophore separation such that CFP–YFP FRET (CYFRET) occurs only if the two proteins are in very close proximity (<50 Å) (Fig. 1, A and B). Thus, CYFRET can be used to measure protein binding in living cells (Tsien, 1998; Miyawaki and Tsien, 2000). However, the majority of studies in living cells have focused on the regulation of intramolecular FRET for CFP–YFP indicator proteins reported as a ratio of YFP to CFP emission during CFP excitation. This ratio is a very sensitive indicator of changes in FRET because acceptor and donor emission have an inverse relationship due to CFP quenching upon FRET with YFP (Miyawaki and Tsien, 2000; Nagai et al., 2000; Zhang et al., 2001). However, CYFRET ratio images are not useful for protein–protein interactions in cells with uncertain stoichiometries due to variable CFP and YFP expression in transient transfections. Recently, a technique using image subtraction, called micro-FRET, that is not subject to these limitations has been shown to produce high-resolution sensitized FRET images showing protein complexes in live cells (Gordon et al., 1998; Sorkin et al., 2000). Thus, we have chosen to use micro-FRET to study PKA and CaN anchoring to AKAP79 in live cells. To independently confirm FRET measured by this method, we have also performed parallel YFP acceptor photobleaching measurement of FRET CFP donor quenching in fixed cells (Miyawaki and Tsien, 2000).


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 FRET microscopy system for studying AKAP79 protein–protein interactions in living cells. (A) Model showing no FRET between A–CFP and B–YFP tagged proteins in the absence of binding. (B) CFP to YFP FRET (CYFRET) resulting in increased YFP acceptor emission and quenching of CFP donor emission seen when A–CFP and B–YFP tagged proteins are bound within 50 Å of each other. (C) CFP- and YFP-tagged (1) AKAP79 WT, (2) ΔPKA, (3) ΔCaN, and (4) (321–360) constructs used for CYFRET imaging of AKAP79 binding to CFP- and YFP-tagged (D) CaNAα and (E) PKA-RIIα. (F) (1) AKAP18(1–16) and (2) AKAP18(1–16)–AKAP79(321–360) hybrid CFP and YFP fusion proteins for CYFRET imaging of CaNA binding. AKAP79 anchors the CaNA,B holoenzyme through binding the catalytic CaNA subunit (Coghlan et al., 1995; Kashishian et al., 1998) and anchors the PKA heterotetrameric R2C2 holoenzyme through binding a surface created by dimerization of the of NH2 terminus of the regulatory subunit (RIIα/β) (Newlon et al., 2001). The NH2 and COOH termini of each protein are indicated by N and C or numbers starting with 1 at the NH2 terminus. The AKAP79 CaN (315–360, pink) and PKA (388–413, red) binding sites are indicated as are the AKAP79 (1–153, A, B, and C poly-basic, blue) and AKAP18 (1–16, NH2-terminal Gly-myristoylated, dual Cys-palmitoylated) membrane targeting domains.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172743&req=5

fig1: CFP/YFP FRET microscopy system for studying AKAP79 protein–protein interactions in living cells. (A) Model showing no FRET between A–CFP and B–YFP tagged proteins in the absence of binding. (B) CFP to YFP FRET (CYFRET) resulting in increased YFP acceptor emission and quenching of CFP donor emission seen when A–CFP and B–YFP tagged proteins are bound within 50 Å of each other. (C) CFP- and YFP-tagged (1) AKAP79 WT, (2) ΔPKA, (3) ΔCaN, and (4) (321–360) constructs used for CYFRET imaging of AKAP79 binding to CFP- and YFP-tagged (D) CaNAα and (E) PKA-RIIα. (F) (1) AKAP18(1–16) and (2) AKAP18(1–16)–AKAP79(321–360) hybrid CFP and YFP fusion proteins for CYFRET imaging of CaNA binding. AKAP79 anchors the CaNA,B holoenzyme through binding the catalytic CaNA subunit (Coghlan et al., 1995; Kashishian et al., 1998) and anchors the PKA heterotetrameric R2C2 holoenzyme through binding a surface created by dimerization of the of NH2 terminus of the regulatory subunit (RIIα/β) (Newlon et al., 2001). The NH2 and COOH termini of each protein are indicated by N and C or numbers starting with 1 at the NH2 terminus. The AKAP79 CaN (315–360, pink) and PKA (388–413, red) binding sites are indicated as are the AKAP79 (1–153, A, B, and C poly-basic, blue) and AKAP18 (1–16, NH2-terminal Gly-myristoylated, dual Cys-palmitoylated) membrane targeting domains.
Mentions: Using GFP as an expression tag to study protein targeting has become an indispensable tool of modern cell biology (Tsien, 1998). CFP and YFP variants of GFP have been developed that can be imaged separately in the same cell. In addition, energy can be transferred from an excited CFP donor to YFP acceptor through a nonradiative dipole–dipole interaction called FRET. There is a strong inverse relationship between FRET and chromophore separation such that CFP–YFP FRET (CYFRET) occurs only if the two proteins are in very close proximity (<50 Å) (Fig. 1, A and B). Thus, CYFRET can be used to measure protein binding in living cells (Tsien, 1998; Miyawaki and Tsien, 2000). However, the majority of studies in living cells have focused on the regulation of intramolecular FRET for CFP–YFP indicator proteins reported as a ratio of YFP to CFP emission during CFP excitation. This ratio is a very sensitive indicator of changes in FRET because acceptor and donor emission have an inverse relationship due to CFP quenching upon FRET with YFP (Miyawaki and Tsien, 2000; Nagai et al., 2000; Zhang et al., 2001). However, CYFRET ratio images are not useful for protein–protein interactions in cells with uncertain stoichiometries due to variable CFP and YFP expression in transient transfections. Recently, a technique using image subtraction, called micro-FRET, that is not subject to these limitations has been shown to produce high-resolution sensitized FRET images showing protein complexes in live cells (Gordon et al., 1998; Sorkin et al., 2000). Thus, we have chosen to use micro-FRET to study PKA and CaN anchoring to AKAP79 in live cells. To independently confirm FRET measured by this method, we have also performed parallel YFP acceptor photobleaching measurement of FRET CFP donor quenching in fixed cells (Miyawaki and Tsien, 2000).

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