Localization of cyclic AMP (cAMP)-dependent protein kinase (PKA) by A kinase-anchoring proteins (AKAPs) restricts the action of this broad specificity kinase. The high-resolution crystal structures of the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA both in the apo state and in complex with the high-affinity anchoring peptide AKAP-IS explain the molecular basis for AKAP-regulatory subunit recognition. AKAP-IS folds into an amphipathic alpha helix that engages an essentially preformed shallow groove on the surface of the RII dimer D/D domains. Conserved AKAP aliphatic residues dominate interactions to RII at the predominantly hydrophobic interface, whereas polar residues are important in conferring R subunit isoform specificity. Using a peptide screening approach, we have developed SuperAKAP-IS, a peptide that is 10,000-fold more selective for the RII isoform relative to RI and can be used to assess the impact of PKA isoform-selective anchoring on cAMP-responsive events inside cells.

M-type (KCNQ2/3) potassium channels are suppressed by activation of G q/11 -coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(ΔA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(ΔA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.The M-current is a low-threshold, slowly activating potassium current that exerts negative control over neuronal excitability. Activation of G q/11 -coupled receptors suppresses the Mcurrent, creating a slow excitatory postsynaptic potential, enhancing excitability and reducing spike-frequency adaptation 1,2 . The M-type K + channel is a promising therapeutic target, as the channel blocker linopirdine acts as a cognition enhancer 3,4 , and the channel activator retigabine functions as an anticonvulsant 5,6 . M-type channels are heteromeric complexes of certain KCNQ-family potassium channel subunits (KCNQ2-5) [7][8][9][10] . KCNQ2 and KCNQ3 were the first members of this family identified as M-channel forming subunits 7 . The KCNQ3 subunit is a core component that co-assembles with KCNQ2, KCNQ4 and KCNQ5 to form functional M-type channels 10 . © 2003 Nature Publishing GroupCorrespondence should be addressed to N.H. (hoshin@ohsu.edu). Note: Supplementary information is available on the Nature Neuroscience website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2014 March 04. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAlthough the subunits that form M-type K + channels have been identified, the molecular details of the signaling pathways that lead to suppression of the M-currents upon receptor stimulation have not yet been fully defined 2,11 . We know that inhibition results from activation of G proteins of the G q/11 family, with the α-subunit as the active moiety 12,13 , and that a 'diffusible' messenger is involved. That is, the receptor/G protein complex can be physically remote from the channel 14,15 . Thus, closure most likely results from some product of phospholipase C activity. M-type channels can be closed by raising intracellular calcium 16 , and there is evidence that this might be a 'second messenger' for bradykinin 17 and nucleotides 18 , but not...

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.

Specificity in cell signalling can be influenced by the targeting of different enzyme combinations to substrates. The A-kinase anchoring protein AKAP79/150 is a multivalent scaffolding protein that coordinates the subcellular localization of second-messenger-regulated enzymes, such as protein kinase A, protein kinase C and protein phosphatase 2B. We developed a new strategy that combines RNA interference of the endogenous protein with a protocol that selects cells that have been rescued with AKAP79/150 forms that are unable to anchor selected enzymes. Using this approach, we show that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type glutamate receptors and M-type potassium channels. Utilization of distinct enzyme combinations in this manner provides a means to expand the repertoire of cellular events that the same AKAP modulates.Cellular regulation must be accomplished through the synchronized actions of a limited number of gene products, as the number of mammalian genes that are required to sustain life is significantly less than was originally anticipated 1,2 . Signal-transduction pathways are created when enzymes, often with broad substrate specificities, act sequentially to evoke cellular responses 3 . Restricting the subcellular localization of these enzymes with scaffolding proteins contributes to the fidelity of each response 4 . Prototypic examples of these are the A-kinase anchoring proteins (AKAPs) that target the cyclic-AMP-dependent protein kinase (protein kinase A, PKA) and other enzymes to defined subcellular locations 5 .AKAP signalling complexes often include signal-transduction and signal-termination enzymes to regulate the forward and backward steps of a given process. The notion of multivalent anchoring proteins was first proposed for the AKAP79 family, which consists of a group of three structurally similar orthologues: human AKAP79, murine AKAP150 and bovine AKAP75 (ref. 6). These AKAPs contain binding sites for PKA, the calcium/ phospholipid-dependent kinase (protein kinase C, PKC) and the calcium/calmodulindependent phosphatase (protein phosphatase 2B, PP2B) 7 . They are tethered to the inner face of the plasma membrane, where they can respond to the generation of second messengers, such as cAMP, calcium and phospholipid 7 . Functional studies have shown that this AKAP family controls the phosphorylation status and action of several ion channels, including AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors, L-type calcium channels, aquaporin water channel, and M-type potassium channels [8][9][10] . One theory that accounts for this diversity of action is that unique combinations of anchored enzymes are recruited to individual ion channels. We tested this hypothesis in cells in which the endogenous anchoring protein was silenced and replaced with AKAP forms that were unable to anchor selected binding partners. Results Functional characterization of AKAP79-depleted cellsPl...

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate contextdependent neuronal signaling events in vivo.AMPA ͉ behavior ͉ KCNQ ͉ knockout S ophisticated systems have evolved to manage the spatial and temporal organization of signal transduction pathways. AKinase Anchoring Proteins (AKAPs) target various protein kinases and phosphatases to subcellular environments where they control the phosphorylation state of neighboring substrates (1). Movement of enzymes in and out of multiprotein complexes contributes to the temporal regulation of signaling. Hence genetic manipulation of AKAP expression impacts the specificity and magnitude of cellular regulation within the context of the whole organism. This is particularly evident in the central nervous system where the elongated and branched morphology of neurons creates many intracellular compartments where AKAPs synchronize neuronal events (2-4).AKAP79/150 is a family of three orthologs (human AKAP79, murine AKAP150, and bovine AKAP75) each initially defined on the basis of its ability to tether the type II PKA holoenzyme (4, 5). Additional binding partners were subsequently identified including PP2B and PKCs (6, 7). Thus, AKAP79/150 complexes can to respond to intracellular second messengers such as cAMP, calcium and phospholipids (7). Furthermore, the simultaneous anchoring of signal transduction and signal termination enzymes influences both forward and backward steps of a cellular event. For example, AKAP79/150 complexes can influence the phosphorylation and action of transmembrane proteins including G protein coupled receptors and adenylyl cyclases (8, 9). Loss of AKAP79/150 from heart cells contributes to the onset of angiotensin II-induced hypertension (10). Electrophysiological approaches have established a role for AKAP79...

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Here, we provide evidence for interactions between AKAP79/150 and ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production.

Control of specificity in cAMP signaling is achieved by A-kinase anchoring proteins (AKAPs), which assemble cAMP effectors such as protein kinase A (PKA) into multiprotein signaling complexes in the cell. AKAPs tether the PKA holoenzymes at subcellular locations to favor the phosphorylation of selected substrates. PKA anchoring is mediated by an amphipathic helix of 14 -18 residues on each AKAP that binds to the R subunit dimer of the PKA holoenzymes. Using a combination of bioinformatics and peptide array screening, we have developed a high affinity-binding peptide called RIAD (RI anchoring disruptor) with >1000-fold selectivity for type I PKA over type II PKA. Cell-soluble RIAD selectively uncouples cAMP-mediated inhibition of T cell function and inhibits progesterone synthesis at the mitochondria in steroid-producing cells. This study suggests that these processes are controlled by the type I PKA holoenzyme and that RIAD can be used as a tool to define anchored type I PKA signaling events.The cAMP signaling pathway synchronizes a variety of physiological responses including cell proliferation and differentiation, microtubule dynamics, reproductive function, modulation of immune responses, and steroidogenesis (1-3). Many of these responses require activation of the cAMP-dependent protein kinase (PKA).3 The dormant PKA holoenzyme is a heterotetramer composed of two catalytic (C) subunits held in an inactive conformation by a regulatory (R) subunit dimer. Upon activation with cAMP, the C subunits are released from their interaction with the R subunit dimer and are free to phosphorylate a plethora of target substrates. Individual cells express a range of PKA isozymes differing in R (RI␣, RI␤, RII␣, RII␤) and C (C␣, C␤, C␥) subunit composition. The type I and type II PKA isozymes, which are classified on the basis of their R subunit dimer, possess slightly different physical and biological properties including a differential sensitivity to cAMP.Spatiotemporal regulation of PKA phosphorylation events is facilitated by A-kinase anchoring proteins (AKAPs). This family of structurally diverse but functionally related proteins act as molecular scaffolds to cluster PKA with specific substrates and signal termination enzymes such as phosphatases (4, 5) and cAMP-specific phosphodiesterases (6 -9). The number of AKAPs has been estimated to more than 75, of which ϳ50 have been identified to date (3, 10). Although the majority of AKAPs described to date bind type II PKA via the RII regulatory subunit, several dual function AKAPs are capable of interacting with both the type I and the type II PKA holoenzyme (11-15). There are also examples of anchoring proteins such as AKAPce (16), PAP7 (17), and merlin (18) that selectively interact with the RI subunit of PKA.The molecular basis for PKA anchoring is the interaction of an amphipathic helical motif of 14 -18 residues on the AKAP with a hydrophobic groove in the N-terminal dimerization domain of the R subunit. High affinity peptides mimicking the amphipathic helix bind to the R...

Compartmentalization of the cAMP-dependent protein kinase (PKA) is coordinated through association with A-kinase anchoring proteins (AKAPs). A defining characteristic of most AKAPs is a 14-to 18-aa sequence that binds to the regulatory subunits (RI or RII) of the kinase. Cellular delivery of peptides to these regions disrupts PKA anchoring and has been used to delineate a physiological role for AKAPs in the facilitation of certain cAMP-responsive events. Here, we describe a bioinformatic approach that yields an RIIselective peptide, called AKAP-in silico (AKAP-IS), that binds RII with a Kd of 0.4 nM and binds RI with a Kd of 277 nM. AKAP-IS associates with the type II PKA holoenzyme inside cells and displaces the kinase from natural anchoring sites. Electrophysiological recordings indicate that perfusion of AKAP-IS evokes a more rapid and complete attenuation of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents than previously described anchoring inhibitor peptides. Thus, computerbased and peptide array screening approaches have generated a reagent that binds PKA with higher affinity than previously described AKAPs.