AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.     

A macromolecular trafficking complex composed of β2-adrenergic receptors,A-Kinase Anchoring Proteins and L-type calcium channels

Abstract

Sympathetic modulation of cardiac L-type calcium channels is an important mechanism for regulating heart rate and cardiac contractility. At the molecular level, activation of β-adrenergic receptors (βAR) increases calcium influx into cardiac myocytes by activating protein kinase A (PKA), leading to subsequent phosphorylation of L-type calcium channels. In the case of the β₂AR, this process is facilitated by the presence of A-Kinase Anchoring Proteins (AKAPs) that serve as scaffolding proteins for the L-type calcium channel and the β₂AR complex. Our work has shown that, in addition to facilitating PKA phosphorylation of the channel, AKAPs also promote an increase in the Cav1.2 channel surface expression. Here we review the molecular mechanisms of β₂AR/AKAP/L-type channel interactions and trafficking.     

Targeting of an A kinase-anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1

Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoring proteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathione S-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites.     

The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons.

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activity of class C L-type Ca(2+) channels which are clustered at postsynaptic sites and are important regulators of neuronal functions. We investigated a possible mechanism that could ensure rapid and efficient phosphorylation of these channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PKA and target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation contain an endogenous kinase that phosphorylates kemptide, a classic PKA substrate peptide, and also the main phosphorylation site for PKA in the pore-forming alpha(1) subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PKA inhibitory peptide PKI(5-24) and stimulated by cAMP. Physical association of the catalytic C subunit of PKA with the immunoisolated class C channel complex was confirmed by immunoblotting. A direct protein overlay binding assay performed with (32)P-labeled RIIbeta revealed a prominent AKAP with an M(r) of 280,000 in class C channel complexes. The protein was identified by immunoblotting as the microtubule-associated protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules. In vitro experiments show that MAP2B can directly bind to the alpha(1) subunit of the class C channel. Our findings indicate that PKA is an integral part of neuronal class C L-type Ca(2+) channels and suggest that the AKAP MAP2B may mediate this interaction. Neither PKA nor MAP2B were detected in immunoprecipitates of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamate receptors or class B N-type Ca(2+) channels. Accordingly, MAP2B docked at class C Ca(2+) channels may be important for recruiting PKA to postsynaptic sites.     

A novel leucine zipper targets AKAP15 and cyclic AMP-dependent protein kinase to the C terminus of the skeletal muscle Ca2+ channel and modulates its function.

In skeletal muscle, voltage-dependent potentiation of L-type Ca(2+) channel (Ca(V)1.1) activity requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 targets PKA to L-type Ca(2+) channels has not been elucidated. Here we report that AKAP15 directly interacts with the C-terminal domain of the alpha(1) subunit of Ca(V)1.1 via a leucine zipper (LZ) motif. Disruption of the LZ interaction effectively inhibits voltage-dependent potentiation of L-type Ca(2+) channels in skeletal muscle cells. Our results reveal a novel mechanism whereby anchoring of PKA to Ca(2+) channels via LZ interactions ensures rapid and efficient phosphorylation of Ca(2+) channels in response to local signals such as cAMP and depolarization.     

A novel lipid-anchored A-kinase Anchoring Protein facilitates cAMP-responsive membrane events.

Compartmentalization of protein kinases with substrates is a mechanism that may promote specificity of intracellular phosphorylation events. We have cloned a low-molecular weight A-kinase Anchoring Protein, called AKAP18, which targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel. Membrane anchoring is mediated by the first 10 amino acids of AKAP18, and involves residues Gly1, Cys4 and Cys5 which are lipid-modified through myristoylation and dual palmitoylation, respectively. Transient transfection of AKAP18 into HEK-293 cells expressing the cardiac L-type Ca2+ channel promoted a 34 9% increase in cAMP-responsive Ca2+ currents. In contrast, a targeting-deficient mutant of AKAP18 had no effect on Ca2+ currents in response to the application of a cAMP analog. Further studies demonstrate that AKAP18 facilitates GLP-1-mediated insulin secretion in a pancreatic beta cell line (RINm5F), suggesting that membrane anchoring of the kinase participates in physiologically relevant cAMP-responsive events that may involve ion channel activation.     

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α_1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β₃ subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.     

Mutations in AKAP5 Disrupt Dendritic Signaling Complexes and Lead to Electrophysiological and Behavioral Phenotypes in Mice

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIα or RIIβ as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and β-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.     

A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels

Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K(+) current (I (Kr)), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I (Kr) by cAMP.     

Cypher/ZASP Is a Novel A-kinase Anchoring Protein

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser²⁶⁵ and Ser²⁹⁶ by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser¹⁹²⁸ induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.     

Imaging kinase--AKAP79--phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. 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. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton-localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 A of each other, thus providing the first direct evidence of a ternary kinase-scaffold-phosphatase complex in living cells. 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. 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.     

Alternative splicing regulates the subcellular localization of A-kinase anchoring protein 18 isoforms

The cAMP-dependent protein kinase (PKA) is localized to specific subcellular compartments by association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Recently, AKAP18, a low molecular weight plasma membrane AKAP that facilitates PKA-mediated phosphorylation of the L-type Ca(2+) channel, was cloned. We now report the cloning of two additional isoforms of AKAP18, which we have designated AKAP18beta and AKAP18gamma, that arise from alternative mRNA splicing. The AKAP18 isoforms share a common R subunit binding site, but have distinct targeting domains. The original AKAP18 (renamed AKAP18alpha) and AKAP18beta target the plasma membrane when expressed in HEK-293 cells, while AKAP18gamma is cytosolic. When expressed in epithelial cells, AKAP18alpha is targeted to lateral membranes, whereas AKAP18beta is accumulated at the apical membrane. A 23-amino acid insert, following the plasma membrane targeting domain, facilitates the association of AKAP18beta with the apical membrane. The data suggest that AKAP18 isoforms are differentially targeted to modulate distinct intracellular signaling events. Furthermore, the data suggest that plasma membrane AKAPs may be targeted to subdomains of the cell surface, adding additional specificity in intracellular signaling.     

Association of an A-kinase-anchoring protein signaling scaffold with cadherin adhesion molecules in neurons and epithelial cells

A-kinase-anchoring protein (AKAP) 79/150 organizes a scaffold of cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and protein phosphatase 2B/calcineurin that regulates phosphorylation pathways underlying neuronal long-term potentiation and long-term depression (LTD) synaptic plasticity. AKAP79/150 postsynaptic targeting requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here, we report a novel interaction of these domains with cadherin adhesion molecules that are linked to actin through beta-catenin (beta-cat) at neuronal synapses and epithelial adherens junctions. Mapping the AKAP binding site in cadherins identified overlap with beta-cat binding; however, no competition between AKAP and beta-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with beta-cat and cadherins in epithelial cell lateral membranes, and beta-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion and actin polymerization redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in LTD that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity.     

The Role of Auxiliary Subunits for the Functional Diversity of Voltage‐Gated Calcium Channels

Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α₁ subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein–protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein–protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. J. Cell. Physiol. 999: 00–00, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2019–2031, 2015. © 2015 Wiley Periodicals, Inc.     

Modulation of calcium-dependent inactivation of L-type Ca2+ channels via β-adrenergic signaling in thalamocortical relay neurons

Neuronal high-voltage-activated (HVA) Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we have shown that β-adrenergic receptor (βAR) stimulation inhibits CDI in rat thalamocortical (TC) relay neurons. This effect can be blocked by inhibition of cAMP-dependent protein kinase (PKA) with a cell-permeable inhibitor (myristoylated protein kinase inhibitor-(14-22)-amide) or A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide, suggesting a critical role of these molecules downstream of the receptor. Moreover, inhibition of protein phosphatases (PP) with okadaic acid revealed the involvement of phosphorylation events in modulation of CDI after βAR stimulation. Double fluorescence immunocytochemistry and pull down experiments further support the idea that modulation of CDI in TC neurons via βAR stimulation requires a protein complex consisting of Ca(V)1.2, PKA and proteins from the AKAP family. All together our data suggest that AKAPs mediate targeting of PKA to L-type Ca(2+) channels allowing their phosphorylation and thereby modulation of CDI.     

Regulation of neuronal PKA signaling through AKAP targeting dynamics

Central to organization of signaling pathways are scaffolding, anchoring and adaptor proteins that mediate localized assembly of multi-protein complexes containing receptors, second messenger-generating enzymes, kinases, phosphatases, and substrates. At the postsynaptic density (PSD) of excitatory synapses, AMPA (AMPAR) and NMDA (NMDAR) glutamate receptors are linked to signaling proteins, the actin cytoskeleton, and synaptic adhesion molecules on dendritic spines through a network of scaffolding proteins that may play important roles regulating synaptic structure and receptor functions in synaptic plasticity underlying learning and memory. AMPARs are rapidly recruited to dendritic spines through NMDAR activation during induction of long-term potentiation (LTP) through pathways that also increase the size and F-actin content of spines. Phosphorylation of AMPAR-GluR1 subunits by the cAMP-dependent protein kinase (PKA) helps stabilize AMPARs recruited during LTP. In contrast, induction of long-term depression (LTD) leads to rapid calcineurin-protein phosphatase 2B (CaN) mediated dephosphorylation of PKA-phosphorylated GluR1 receptors, endocytic removal of AMPAR from synapses, and a reduction in spine size. However, mechanisms for coordinately regulating AMPAR localization, phosphorylation, and synaptic structure by PKA and CaN are not well understood. A kinase-anchoring protein (AKAP) 79/150 is a PKA- and CaN-anchoring protein that is linked to NMDARs and AMPARs through PSD-95 and SAP97 membrane-associated guanylate kinase (MAGUK) scaffolds. Importantly, disruption of PKA-anchoring in neurons and functional analysis of GluR1-MAGUK-AKAP79 complexes in heterologous cells suggests that AKAP79/150-anchored PKA and CaN may regulate AMPARs in LTD. In the work presented at the "First International Meeting on Anchored cAMP Signaling Pathways" (Berlin-Buch, Germany, October 15-16, 2005), we demonstrate that AKAP79/150 is targeted to dendritic spines by an N-terminal basic region that binds phosphatidylinositol-4,5-bisphosphate (PIP(2)), F-actin, and actin-linked cadherin adhesion molecules. Thus, anchoring of PKA and CaN as well as physical linkage of the AKAP to both cadherin-cytoskeletal and MAGUK-receptor complexes could play roles in coordinating changes in synaptic structure and receptor signaling functions underlying plasticity. Importantly, we provide evidence showing that NMDAR-CaN signaling pathways implicated in AMPAR regulation during LTD lead to a disruption of AKAP79/150 interactions with actin, MAGUKs, and cadherins and lead to a loss of the AKAP and anchored PKA from postsynapses. Our studies thus far indicate that this AKAP79/150 translocation depends on activation of CaN, F-actin reorganization, and possibly Ca(2+)-CaM binding to the N-terminal basic regions. Importantly, this tranlocation of the AKAP79/150-PKA complex from spines may shift the balance of PKA kinase and CaN/PP1 phosphatase activity at the postsynapse in favor of the phosphatases. This loss of PKA could then promote actions of CaN and PP1 during induction of LTD including maintaining AMPAR dephosphorylation, promoting AMPAR endocytosis, and preventing AMPAR recycling. Overall, these findings challenge the accepted notion that AKAPs are static anchors that position signaling proteins near fixed target substrates and instead suggest that AKAPs can function in more dynamic manners to regulate local signaling events.