Spatiotemporal specificity of cAMP action is best explained by targeting protein kinase A (PKA) to its substrates by A-kinase-anchoring proteins (AKAPs). At synapses in the brain, AKAP79/150 incorporates PKA and other regulatory enzymes into signal transduction networks that include ␤-adrenergic receptors, ␣-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA), and N-methyl-D-aspartic acid receptors. We previously showed that AKAP79/150 clusters PKA with type 5 adenylyl cyclase (AC5) to assemble a negative feedback loop in which the anchored kinase phosphorylates AC5 to dynamically suppress cAMP synthesis. We now show that AKAP79 can associate with multiple AC isoforms. The N-terminal regions of AC5, -6, and -9 mediate this protein-protein interaction. Localized activation of PKA 2 triggers a plethora of intracellular signaling processes (1). Precise control of these phosphorylation events is often achieved by restricted activation of PKA in discrete microenvironments. AKAPs participate in this process by tethering the kinase close to preferred substrates. AKAPs now represent a family of 43 diverse but functionally related proteins that bind the regulatory subunit dimer of the PKA holoenzyme (2).AKAPs have been identified in a range of species, tissues, and cellular compartments. The AKAP79/150 group of anchoring proteins is perhaps the best understood member of this class of signal-organizing proteins. AKAP79/150 consists of three orthologs: bovine AKAP75, human AKAP79, and murine AKAP150. Although originally identified in the postsynaptic densities of neurons, this group of anchoring proteins is also expressed in a variety of other tissues. In addition to binding PKA, AKAP79 has the ability to bind protein phosphatase 2B (3) and protein kinase C (PKC) (4, 5). By organizing these signal transduction and signal termination enzymes in the same location, AKAP79 provides a platform to facilitate the bidirectional control of cAMP-and calcium-mediated signaling events.Although anchoring of PKA with its substrates provides an efficient mechanism for the spatial regulation necessary for selectivity of cAMP signaling, it was not clear how local pools of cAMP are managed. We have shown that AC isoforms can specifically interact with three different AKAP complexes, AKAP79, Yotiao, and mAKAP, to regulate events downstream of cAMP production (6 -8). We have also demonstrated that anchoring of AC5 to an AKAP79/150 complex provides negative feedback on AC5 via PKA phosphorylation of AC5 within the complex (6).Although characterization of the AKAP79-AC5 interaction has shed some light on the advantages gained by localizing different components of cAMP signaling pathways, several key issues remain unresolved. First of all, do other AC isoforms interact with AKAP79 or other anchoring proteins? Secondly, are AC isoforms recruited into larger signaling networks via their protein-protein interactions with AKAP79? AKAP79/150 has been shown to form a multiprotein signaling complex with AMPA and NMDA receptors (9 -11), adhesion mol...

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein ␤ (mAKAP␤) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAP␤ selectively binds type 5 AC in the heart and that mAKAP␤-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAP␤-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAP␤-AC association. Accordingly, disruption of mAKAP␤-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAP␤ complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.The formation of multimolecular protein complexes contributes to the specificity of intracellular signaling pathways, including those regulating cardiac myocyte hypertrophy. The cAMP-dependent protein kinase (PKA) 3 is targeted to specific intracellular domains by protein kinase A-anchoring proteins (AKAPs) that often serve as scaffolding proteins for diverse signaling enzymes (1). In the heart, global disruption of PKA anchoring affects cardiac contractility, while the inhibited expression of individual AKAPs such as mAKAP␤ or AKAPLbc attenuates adrenergic-induced hypertrophy of cultured neonatal myocytes (2-4). We have recently shown that specific AKAPs, namely AKAP79 and Yotiao, bind adenylyl cyclases (AC) (5, 6). However, the functional significance of AC-AKAP complexes has not been demonstrated. mAKAP␤, expressed in striated myocytes, is one of two known splice variants encoded by the single mAKAP (AKAP6) gene (7). We previously published that mAKAP␤ is primarily localized to the outer membrane of the nuclear envelope via direct binding to nesprin-1␣ (4, 8). In cardiac myocytes, mAKAP␤ serves as the scaffold for a multimolecular signaling complex that in addition to PKA includes the ryanodine receptor (RyR2), the protein phosphatases PP2A and calcineurin, phosphodiesterase 4D3 (PDE4D3), exchange protein activated by cAMP (Epac1), ERK5, and MEK5 mitogen-activated protein kinases, molecules implicated in the regulation of cardiac hypertrophy (4, 7-13). mAKAP␤ complexes facilitate crosstalk between MAP kinase, calcium, and cAMP signaling pathways, permitting feedback inhibition of cAMP levels and the dynamic regulation of PKA and ERK5 activity (4, 9 -13). Accordingly, mAKAP␤ RNAi attenuates adrenergic and cytokine-induced hypertrophy of...

Endocytosis of Na + ,K + -ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I A phosphoinositide-3 kinase (PI3K-I A ) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I A , through its p85α subunit-SH3 domain, binds to a proline-rich region in the Na + ,K + -ATPase catalytic α subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na + ,K + -ATPase α subunit and results in increased PI3K-I A activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na + ,K + -ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I A and its activation during Na + ,K + -ATPase endocytosis in response to G protein-coupled receptor signals.