Spatiotemporal regulation of protein kinase A (PKA) activity involves the manipulation of compartmentalized cAMP pools. Now we demonstrate that the muscle-selective A-kinase anchoring protein, mAKAP, maintains a cAMP signaling module, including PKA and the rolipram-inhibited cAMP-speci®c phosphodiesterase (PDE4D3) in heart tissues. Functional analyses indicate that tonic PDE4D3 activity reduces the activity of the anchored PKA holoenzyme, whereas kinase activation stimulates mAKAP-associated phosphodiesterase activity. Disruption of PKA± mAKAP interaction prevents this enhancement of PDE4D3 activity, suggesting that the proximity of both enzymes in the mAKAP signaling complex forms a negative feedback loop to restore basal cAMP levels.

The mechanism by which protein kinase A (PKA) inhibits G␣ q -stimulated phospholipase C activity of the ␤ subclass (PLC␤) is unknown. We present evidence that phosphorylation of PLC␤ 3 by PKA results in inhibition of G␣ q -stimulated PLC␤ 3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLC␤ 3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. Ligand stimulation of seven transmembrane domain receptors coupled to G␣ proteins of the G␣ q or G␣ i subfamilies results in the activation of the respective heterotrimeric G␣␤␥ protein complexes. Free G␣ q or G␤␥ subunits activate PLC␤ 1 isoforms to catalyze the production of IP 3 and diacylglycerol from phosphatidylinositide 4,5-bisphosphate (1-3). PLC␤ 1-4 comprise the currently known mammalian phosphatidylinositide-specific PLC␤ subfamily. Although all PLC␤s are activated by G␣ q , PLC␤ 2 and PLC␤ 3 are also stimulated by G␤␥, primarily released from G␣ i (1).Cross-talk between the G protein-PLC␤ pathway and PKA has been documented in numerous studies (4 -13). Although it is generally agreed that G protein-activated PLC␤ activity can be inhibited by PKA (4 -11), PKA can enhance the G protein-PLC␤ pathway in some cases (12, 13). Because PKA can inhibit phosphatidylinositide (PI) turnover activated by both G␣ q (4 -8) and G␣ i (9 -11) coupled receptors, it may inhibit the stimulation of both G␣ q -and G␤␥-stimulated PLC␤ activity. This notion is further supported by studies with the G protein activators GTP␥S and AlF 4 Ϫ . These two compounds nonselectively activate all heterotrimeric G proteins and generate free G␣ and G␤␥ subunits that can stimulate PLC␤s. PKA inhibition of PI turnover initiated by GTP␥S or AlF 4 Ϫ (5, 8, 14, 15) is consistent with the inhibition of G␣q-as well as G␤␥-stimulated PLC␤ activity. In addition, this phenomenon also suggests that the PKA effect is distal to receptors.Recently, the mechanism for PKA inhibition of G␤␥-stimulated PI turnover has been elucidated. Phosphorylation of PLC␤ 2 by PKA resulted in inhibition of G␤␥-stimulated PI turnover (10). However, in the same study, PKA apparently did not inhibit G␣ 15 -and G␣ 16 -stimulated endogenous PLC␤ (␤ 1 and ␤ 3 ) activity. More recently, Ali et al. (11) have reported phosphorylation of PLC␤ 3 in response to CPT-cAMP treatment in RBL-2H3 cells expressing only PLC␤ 3 . CPT-cAMP inhibited G␤␥-stimulated PLC␤ 3 activated by the G␣ i -coupled formylmethionylleucylphenylalanine receptor but had no effect on PAFstimulated PLC␤ 3 activity, presumably mediated by G␣ q . These studies led to the conclusions that phosphorylation of PLC␤ 2 and PLC␤ 3 by PKA could explain the inhibition of G␤␥-stimulated PI turnover by cAMP (10, 11). However, a biochemical mechanism for the inhibition by PKA of G␣ q -stimulated PLC␤ activity observed in several systems remains to be clarified. In this study, we present evidence that phosphorylation of PLC␤ 3 Ser 1105 by PKA results in d...

Compartmentalization of protein kinases and phosphatases with substrates is a means to increase the efficacy of signal transduction events. The A-kinase anchoring protein, AKAP79, is a multivalent anchoring protein that maintains the cAMP-dependent protein kinase, protein kinase C, and protein phosphatase-2B (PP2B/calcineurin) at the postsynaptic membrane of excitatory synapses where it is recruited into complexes with Nmethyl-D-aspartic acid or ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-subtype glutamate receptors. We have used cellular targeting of AKAP79 truncation and deletion mutants as an assay to map the PP2B-binding site on AKAP79. We demonstrate that residues 315-360 are necessary and sufficient for AKAP79-PP2B anchoring in cells. Multiple determinants contained within this region bind directly to the A subunit of PP2B and inhibit phosphatase activity. Peptides spanning the 315-360 region of AKAP79 can antagonize PP2B anchoring in vitro and targeting in transfected cells. Electrophysiological experiments further emphasize this point by demonstrating that a peptide encompassing residues 330 -357 of AKAP79 attenuates PP2B-dependent down-regulation of GluR1 receptor currents when perfused into HEK293 cells. We propose that the structural features of this AKAP79-PP2B-binding domain may share similarities with other proteins that serve to coordinate PP2B localization and activity.The efficient transmission of cellular signals often involves the orientation of signaling proteins in relation to their upstream activators and downstream targets. This is often achieved through association with anchoring and scaffolding proteins that compartmentalize signaling enzymes in distinct subcellular environments (1-3). For example, A-kinase anchoring proteins (AKAPs) 1 bind the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) to localize this broad specificity enzyme to discrete subcellular environments (4, 5). Each AKAP contains a conserved amphipathic helix that binds to the R subunit dimer with high affinity and targeting domains that direct the PKA-AKAP complex to specific subcellular compartments (6, 7). A likely consequence of these proteinprotein interactions is that AKAP-PKA complexes are maintained in the vicinity of selected phosphoproteins and substrates for the kinase. Another important role of AKAPs is to serve as scaffolds for the assembly of multiprotein complexes that include PKA, other protein kinases, phosphodiesterases, and a variety of protein phosphatases (8). The simultaneous anchoring of kinases and phosphatases provides an efficient means to confer bi-directional control on the phosphorylation status of substrate proteins (9, 10).A number of studies (11) have demonstrated that anchoring of kinases and phosphatases ensures the efficient regulation of ion channels and neurotransmitter receptors. One prominent mediator of this process is the multivalent anchoring protein AKAP79 that anchors PKA, protein kinase C (PKC), and protein phosphatase-2B (PP2B/calcineurin) (12...