Importantly, PKA, CaN, and SAP97 have also been implicated in the regulation of AMPA receptor activity in LTP and LTD ( Hayashi et al., 2000 Lee et al., 2000 Carroll et al., 2001). AKAP79 is recruited to NMDA and AMPA glutamate receptors at synapses through binding the MAGUKs PSD-95 and synapse associated protein 97 (SAP97), respectively, and anchored PKA and CaN in these complexes participate in the regulation of AMPA receptor phosphorylation ( Rosenmund et al., 1994 Colledge et al., 2000 Dell'Acqua et al., 2002 Tavalin et al., 2002). In hippocampal neurons, AKAP79, PKA, and CaN are localized on postsynaptic dendritic spines with F-actin and PSD-95 family membrane-associated guanylate kinase (MAGUK) PDZ scaffolds ( Gomez et al., 2002). AKAP79 is a membrane/cortical cytoskeleton–targeted anchoring protein that binds the cAMP-dependent protein kinase (PKA) regulatory (R) subunits, PKC, and protein phosphatase 2B/calcineurin catalytic A subunit (CaNA) ( Coghlan et al., 1995 Klauck et al., 1996 Dell'Acqua et al., 1998 Gomez et al., 2002). One prototypic signaling scaffold that might have important functions in synaptic plasticity is A-kinase anchoring protein (AKAP) 79 (humanAKAP79/ratAKAP150) ( Bregman et al., 1989 Carr et al., 1992). 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. 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. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. 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. Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction.
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