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...

mAKAP (muscle-selective A-kinase-anchoring protein) co-ordinates a cAMP-sensitive negative-feedback loop comprising PKA (cAMP-dependent protein kinase) and the cAMP-selective PDE4D3 (phosphodiesterase 4D3). In vitro and cellular experiments demonstrate that PKA-phosphorylation of PDE4D3 on Ser-13 increases the affinity of PDE4D3 for mAKAP. Our data suggest that activation of mAKAP-anchored PKA enhances the recruitment of PDE4D3, allowing for quicker signal termination.

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Compartmentalization of kinases and phosphatases is a key determinant in the specificity of second messenger؊ mediated signaling events. Localization of the cAMPdependent protein kinase (PKA) and other signaling enzymes is mediated by interaction with A-kinase anchoring proteins (AKAPs). This study focused on recent advances that further our understanding of AKAPs, with particular emphasis on the bidirectional regulation of signaling events by AKAP signaling complexes and their contribution to the control of actin reorganization events. Diabetes 51 (Suppl. 3):S385-S388, 2002 E xtracellular signals, such as hormones, neurotransmitters, and growth factors, regulate a wide variety of cellular activities, including ion channel modulation, neuronal excitation, cell growth, cell differentiation, and insulin secretion events (1). Intracellular transduction systems receive these signals via receptors and transmit them quickly and precisely, resulting in the amplification of specific biological responses. However, cells often are exposed to several messengers simultaneously, and maintaining the fidelity of these networks is crucial in eliciting the appropriate physiological response. Doing so requires the accurate selection of effector molecules for regulated activation and deactivation, often by phosphorylation and dephosphorylation events. A principal strategy in achieving this selection specificity is compartmentalization of signaling enzymes (2-4). This study highlights the most recent advances in our understanding of compartmentalization of multivalent signaling complexes by A-kinase anchoring proteins (AKAPs) and the functional consequences they mediate. CYCLIC AMP-DEPENDENT PROTEIN KINASEOne of the best-characterized signaling pathways involves the activation of the cAMP-dependent protein kinase (PKA). PKA is a serine/threonine kinase composed of two catalytic (C) subunits that are held in an inactive state by association with a regulatory (R) subunit dimer (5-8). The catalytic subunits are expressed from three different genes-C␣, C␤, and C␥-whereas the R subunits are expressed from four different genes-RI␣, RI␤, RII␣, and RII␤ (9 -12). The R subunit is a modular polypeptide containing an NH 2 -terminal dimerization domain, an autophosphorylation site that serves as a principal contact site for the C subunit, and two cAMP binding sites. Activation of PKA is solely accomplished by the major, diffusible secondary messenger cAMP (13,14). Binding of cAMP to each R subunit relieves the autoinhibitory contact, allowing the C subunits to dissociate (15,16), thereby resulting in phosphorylation of local substrates.Two forms of the heterotetrameric PKA holoenzyme exist: type I (RI␣ and RI␤ dimer) and type II (RII␣ and RII␤ dimer). Type I PKA is predominantly cytoplasmic, whereas type II PKA associates with specific cellular structures and organelles (17). Discrete localization of type II PKA within the cell is chiefly caused by association with nonenzymatic scaffolding AKAPs (3,18,19). This method of regulation ensures tha...

The importance of the localization of protein kinase A (PKA) to the plasma membrane for cAMP-mediated inhibition of phosphatidylinositide turnover was tested in an immortalized pregnant human myometrial (PHM1-41) cell line, and the putative A kinase anchoring protein (AKAP) involved was identified. Preincubation in PHM1-41 cells with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited the ability of oxytocin to stimulate phosphatidylinositide turnover. The addition of a peptide that specifically disrupts interactions of PKA RII subunits with AKAPs (S-Ht31) reversed the effects of these agents, whereas a control peptide was ineffective. The pharmacology of S-Ht31 on this particular membrane event was further characterized. A 10-min incubation with S-Ht31 at a concentration of 1 microM completely reversed the inhibitory effect of relaxin on phosphatidylinositide turnover. S-Ht31 inhibited cAMP-stimulated PKA activity in PHM1-41 cell plasma membranes and decreased the concentration of PKA. Overlay analysis detected a single AKAP of approximately 86 kDa associated with the plasma membrane of PHM1-41 cells, suggesting that the association of PKA with this AKAP is important for the cAMP inhibitory mechanism. The mol wt of this AKAP was similar to that of an AKAP associated with the plasma membrane in the human brain, AKAP79. Antibodies against AKAP79 recognized a band at 86 kDa in purified plasma membranes from the PHM1-41 cells, indicating similar determinants in these proteins. These data suggest that PKA is anchored to the myometrial plasma membrane through association with an AKAP similar to AKAP79, and that this anchoring is required for the cAMP-mediated inhibition of phosphatidylinositide turnover in PHM1-41 cells.

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