A-kinase Anchoring Proteins Research Articles

Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates

Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.

Subcellular Localization and Functional Characterization of cAMP‐dependent Protein Kinase A Isoforms: Painting Specificity by Mosaic Brain Mapping

cAMP dependent Protein kinase (PKA) plays a critical role in numerous neuronal functions. Specificity in PKA signaling is achieved in part by the four functionally non‐redundant regulatory (R) subunits. The inactive holoenzyme has a dimeric R‐subunit bound to two Catalytic (C) subunits. The full‐length holoenzyme crystal structure allowed me to understand how isoform‐specific assembly can create distinct holoenzyme structures that each defines its allosteric regulation. High‐resolution large‐scale mosaic images provide global views of brain sections and allow identification of subcellular features. Analysis of multiple regions demonstrates that the R‐isoforms are concentrated within discrete regions and express unique patterns of subcellular localization. Using correlated light and electron microscopy I confirmed mitochondrial and nuclear localization of RIb isoform, modifying the existing dogma of cAMP‐PKA in the nucleus. To show the functional significance of nuclear localization, I demonstrated that RIb down‐regulation, but not RIIb, decreased CREB phosphorylation in primary neuronal cultures, consistent with deficits observed in RIb mutant mice. Furthermore, I show that a point mutation in the RIb gene, found in patients diagnosed with a neurodegenerative disease, abolishes dimerization while retaining robust interaction with its catalytic subunit. As a consequence, the interaction with A Kinase Anchoring Proteins (AKAPs) is also abolished. Quantitative mass spectroscopy analysis of patient samples identified a set of proteins that were up or down regulated in this disease. This point mutation further emphasizes the importance of precisely controlled PKA isoform subcellular localization as defined by my mosaic maps.

OPA1 in Lipid Metabolism: Function of OPA1 in Lipolysis and Thermogenesis of Adipocytes.

OPA1 (Optic Atrophy 1) is a mitochondrial GTPase known to regulate fission of mitochondria. It was recently also shown to locate on lipid droplets in adipocytes where it functions as an A-kinase anchoring protein (AKAP) that mediates adrenergic control of lipolysis by facilitating PKA phosphorylation of perilipin (Plin1). In brown adipocytes indirect evidence support the notion that OPA1 regulation of fission serves to increase thermogenesis, which thereby contributes to dissipation of energy. In white adipocytes, OPA1 located on lipid droplets serves as a gatekeeper to control lipolysis induced by adrenergic agonists. However, the function of OPA1 in lipolysis and thermogenesis in inducible brown adipocytes (brite/beige cells) remains elusive. Here we discuss the role of OPA1 in lipid metabolism.

Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non‐Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G‐protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non‐lipid raft domains of the plasma membrane, β‐Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non‐lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically‐encoded FRET‐based biosensors targeted to lipid raft and non‐lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non‐lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non‐lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A‐kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains.Support or Funding InformationSupport by: NIH R01 GM107094

SRY‐box 9 (SOX9): A Novel Binding Partner of Cardiac Muscle‐specific A‐Kinase Anchoring Protein (mAKAP) Signalosome

BackgroundBy definition, A‐kinase anchoring proteins (AKAPs) bind to protein kinase A and various other signaling proteins at specific subcellular compartments to regulate downstream signal transduction. Heart specific muscle AKAP (mAKAP) is present on the nuclear envelope of cardiomyocytes and regulates cardiac hypertrophy by forming a signalosome of its binding partners. It was previously reported that hypoxia‐inducible factor 1 alpha (HIF‐1α) physically binds to mAKAP. Also, it was previously published that HIF‐1α physically interacts with SRY‐box 9 (SOX9). Chromatin immunoprecipitation (ChIP) studies showed that HIF‐1α binds to SOX9 promoter region. Interestingly, both mAKAP and SOX9 have been demonstrated to play significant role in cardiac differentiation and endocardial cushion formation, respectively. Hence, we were interested to know whether SOX9 is a part of mAKAP scaffold on the nuclear envelope.ObjectiveThe aim of the current work is to confirm that SOX9 unambiguously interacts with mAKAP; either directly or indirectly.MethodsAs HEK 293T cells express SOX9 endogenously and not mAKAP, we chose these cells for immunoprecipitation (IP) studies. Myc‐DDK tagged human mAKAP cDNA was purchased from Origene and overexpressed in HEK 293T cells using Lipofectamine 2000, according manufacturer's instructions. IP was performed using M2 affinity gels (Sigma) and immunoblotting was performed using mAKAP and SOX9 antibodies. HepG2 cell lysate was used as positive control for SOX9 expression. In another set of experiment, a Coomassie Blue stained gel was used for in‐gel digestion to perform MALDI‐TOF Mass Spectrometry, followed by data analysis.Summary of ResultsIP samples of mAKAP transfected HEK cells displayed SOX9 expression; whereas, untransfected cells did not show expression. mAKAP expression was used as a loading control and remained unchanged in all samples. Mass Spectrometric data validated our findings by demonstrating the expected mass peak for SOX9 in the IP samples.Conclusion and Future ImplicationsSOX9 was identified as a novel binding partner of mAKAP. As mAKAP and SOX9 already have a role in cardiac developmental biology, we suggest that mAKAP‐SOX9 interaction has substantial role in cardiac physiology and pathophysiology. The significance of this mAKAP‐SOX9 interaction is a part of our ongoing investigations.Support or Funding InformationR15HL124458 Scaffolding Protein Interaction Network Regulates Cardiac Cell Signaling

Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System

The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.

Protein kinase A regulates C-terminally truncated CaV 1.2 in Xenopus oocytes: roles of N- and C-termini of the α1C subunit.

β-Adrenergic stimulation enhances Ca2+ entry via L-type CaV 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of CaV 1.2 remain controversial despite extensive research. We show that PKA regulation of CaV 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α1C , is truncated. The PKA upregulation of CaV 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of CaV 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of CaV 1.2 in the heart. β-Adrenergic stimulation enhances Ca2+ currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signalling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 currents by ∼40% (rabbit α1C ) to ∼130% (mouse α1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The CaV β2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α1C .

Role of plasma membrane-associated AKAPs for the regulation of cardiac IK1 current by protein kinase A.

The cardiac IK1 current stabilizes the resting membrane potential of cardiomyocytes. Protein kinase A (PKA) induces an inhibition of IK1 current which strongly promotes focal arrhythmogenesis. The molecular mechanisms underlying this regulation have only partially been elucidated yet. Furthermore, the role of A-kinase anchoring proteins (AKAPs) in this regulation has not been examined to date. The objective of this project was to elucidate the molecular mechanisms underlying the inhibition of IK1 by PKA and to identify novel molecular targets for antiarrhythmic therapy downstream β-adrenoreceptors. Patch clamp and voltage clamp experiments were used to record currents and co-immunoprecipitation, and co-localization experiments were performed to show spatial and functional coupling. Activation of PKA inhibited IK1 current in rat cardiomyocytes. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide inhibitor AKAP-IS. We observed functional and spatial coupling of the plasma membrane-associated AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channel subunits, but not to Kir2.3 channels. In contrast, AKAPyotiao had no functional effect on the PKA regulation of Kir channels. AKAP15 and AKAP79 co-immunoprecipitated with and co-localized to Kir2.1 and Kir2.2 channel subunits in ventricular cardiomyocytes. In this study, we provide evidence for coupling of cardiac Kir2.1 and Kir2.2 subunits with the plasma membrane-bound AKAPs 15 and 79. Cardiac membrane-associated AKAPs are a functionally essential part of the regulatory cascade determining IK1 current function and may be novel molecular targets for antiarrhythmic therapy downstream from β-adrenoreceptors.

AKAP-mediated feedback control of cAMP gradients in developing hippocampal neurons.

Cyclic AMP (cAMP) and protein kinase A (PKA), classical examples of spatially compartmentalized signaling molecules, are critical axon determinants that regulate neuronal polarity and axon formation, yet little is known about micro-compartmentalization of cAMP and PKA signaling and its role in developing neurons. Here, we revealed that cAMP forms a gradient in developing hippocampal neurons, with higher cAMP levels in more distal regions of the axon compared to other regions of the cell. Interestingly, this cAMP gradient changed according to the developmental stage and depended on proper anchoring of PKA by A-kinase anchoring proteins (AKAPs). Disrupting PKA anchoring to AKAPs increased the cAMP gradient in early-stage neurons and led to enhanced axon elongation. Our results provide new evidence for a local negative feedback loop, assembled by AKAPs, for the precise control of a growth-stage-dependent cAMP gradient to ensure proper axon growth.

PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes

PKA Signaling: Linkers, Loops, and Intrinsically Disorder Regions Drive the Dynamic Assembly of Holoenzyme Complexes.The PKA catalytic (C) subunit crystallized in 1991, gave us our first glimpse of the fold that describes all protein kinases. The two lobes that comprise the core are flanked by N-and C-terminal tails, which wrap around both lobes. By capturing all stages of catalysis in crystal structures we appreciate the dynamic nature of these tails and how they undergo order-disorder transitions as part of the catalytic cycle. Ordering the activation loop by a critical phosphate is another fundamental order/disorder transition that is a conserved feature of most protein kinases. These dynamic features allow the kinases to function as highly regulated molecular switches. In the case of PKA, the C-subunit is packaged with regulatory (R) subunit dimers where each chain contains two C-terminal cAMP binding (CNB) domains. The CNB domains are highly dynamic allosteric signaling modules that have been conserved to translate a biological response to cAMP. Although the fundamental features of this domain were elucidated by structures of R-monomers and R:C heterodimers, it required full-length R2C2 tetramers to appreciate the importance of symmetry for PKA signaling. Each R-subunit contains an N-terminal dimerization domain that is also a docking site for scaffold proteins referred to as A Kinase Anchoring Proteins (AKAPs). This domain is joined by a flexible linker to the CNB domains, and embedded within the linker is an inhibitor site that docks to the active site of the C-subunit in the holoenzyme. The order/disorder transitions of the linker drive the assembly of the holoenzymes which each have distinct quaternary structures. The linkers, rich in biological information, are an essential feature of PKA regulation. (Funded by NIH GM34921 and DK54441.)

A protein kinase A-ezrin complex regulates connexin 43 gap junction communication in liver epithelial cells

Communication between adjacent cells can occur via gap junctions (GJ) composed of connexin (Cx) hexamers that allow passage of small molecules. One of the most widely and highly expressed Cxs in human tissues is Cx43, shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane associated protein that can serve as an A-kinase anchoring protein (AKAP) and hold an anchored pool of PKA. Here, we used the liver epithelial cell line IAR20, which expresses Cx43 as the predominant GJ protein, to test the hypothesis that Ezrin may associate with Cx43 in cell types that form stable GJs and serve as an AKAP. Our biochemical and proteomics data indicate that Ezrin associates with Cx43 in epithelial cells. Analyses by confocal immunofluorescence microscopy and proximity ligation assays demonstrate that Ezrin and Cx43 co-localize, together with zonula occludens-1 (ZO-1) and PKA RIα and RIIα, at the cell membrane. Quantitative gap-FRAP experiments show increased GJ intercellular communication after cAMP stimulation. Moreover, loading of cells with the Ht31 peptide that displaces both PKA RIα and RIIα from the AKAP or a peptide that disrupts the Cx43-Ezrin interaction reverts the effect and reduces the level of communication, supporting the hypothesis that in IAR20 cells Ezrin associates with Cx43 (in complex with ZO-1) which places PKA in proximity to Cx43, enabling its phosphorylation and GJ opening.

Structural features of FAP174, a MYCBP-1 orthologue from Chlamydomonas reinhardtii, revealed by computational and experimental analyses

The ciliary MYCBP-1 (FAP174) fromChlamydomonas reinhardtiiis an R2D2 protein and harbors a Dimerization and Docking domain.

A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43–Ezrin Interaction Disruptors Using IncuCyte ZOOM

Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin–protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

Construction and analysis of a human testis/sperm-enriched interaction network: Unraveling the PPP1CC2 interactome

BackgroundPhosphoprotein phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a PPP1CC tissue-specific alternative splice restricted to testicular germ cells and spermatozoa, is essential for spermatogenesis and spermatozoa motility. The key to understand PPP1CC2 regulation lies on the characterization of its interacting partners. MethodsWe construct a testis/sperm-enriched protein interaction network and analyzed the topological properties and biological context of the network. Further the interaction of a potential target for pharmacological intervention was validated in human spermatozoa. ResultsA total of 1778 proteins and 32,187 interactions between them were identified in the testis/sperm-enriched network. The network analysis revealed the members of functional modules that interact more tightly with each other. In the network, PPP1CC was located in the fourth maximum core part (k=41) and had 106 direct interactors. Sixteen PPP1CC interactors were involved in spermatogenesis-related categories. Also, PPP1CC had 50 direct interactors, highly interconnected and many of them part of the network maximum core (k=44), associated with motility-related annotations, including several previously uncharacterized interactors, such as, LMNA, JAK2 and RIPK3. ConclusionsIn this study we integrated tissue-specific protein expression and protein-protein interaction data in order to identify key PPP1CC2 complexes for male reproductive functions.One of the most intriguing interactors was A-kinase anchor protein 4 (AKAP4), a testis-specific protein related to infertility phenotypes and involved in all major motility-related annotations. General significanceWe demonstrated for the first time the interaction between PPP1CC2 and AKAP4 in human spermatozoa and the potential of the complex as contraceptive target.

A-Kinase Anchoring Protein 4 (AKAP4) is an ERK1/2 substrate and a switch molecule between cAMP/PKA and PKC/ERK1/2 in human spermatozoa.

Mammalian spermatozoa undergo capacitation and acrosome reaction in order to fertilize the egg. The PKC-ERK1/2 pathway plays an important role in human spermatozoa motility, capacitation and the acrosome reaction. Here we demonstrate that ERK1/2 phosphorylates proAKAP4 on Thr265 in human spermatozoa in vitro and in vivo. Cyclic AMP (cAMP) had no effect on ERK1/2 activity in human spermatozoa, but stimulated the MAPK in mouse pituitary LβT2 gonadotrope cells. cAMP via PKA attenuates PKC-dependent ERK1/2 activation only in the presence of proAKAP4. St-HT31, which disrupts PKA-regulatory subunit II (PKA-RII) binding to AKAP abrogates the inhibitory effect of cAMP in human spermatozoa and in HEK293T cells expressing proAKAP4. In transfected HEK293T cells, PMA relocated proAKAP4, but not proAKAP4-T265A to the Golgi in an ERK1/2-dependnet manner. Similarly, AKAP4 is localized to the spermatozoa principal piece and is relocated to the mid-piece and the postacrosomal region by PMA. Furthermore, using capacitated sperm we found that cAMP reduced PMA-induced ERK1/2 activation and acrosome reaction. Thus, the physiological role of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways is to regulate capacitation and acrosome reaction.

Regulation of the U3-, U8-, and U13snoRNA Expression by the DEAD Box Proteins Ddx5/Ddx17 with Consequences for Cell Proliferation and Survival.

Small nucleolar RNAs (snoRNAs) in cooperation with their associated proteins (snoRNPs) contribute to the maturation of ribosomal RNA, transfer RNA, and other transcripts. Most snoRNPs mediate chemical base modifications of their RNA substrates, and a few others, like those formed by the C/D snoRNAs U3, U8, and U13, are needed for the structural organization and maturation of primary transcripts. The U3-, U8-, and U13snoRNAs are encoded by autonomous genes, and our knowledge about their expression regulation is limited. In this study, a significant increase in the concentrations of U3-, U8-, and U13snoRNA after a knockdown of DEAD box proteins Ddx5/Ddx17 in HeLa cells is observed. These alterations are shown to be caused by transcriptional suppression mediated by Ddx5/Ddx17 via histone deacetylase 1 in a promoter-dependent way. The biological function of this expression control may be related to the role of Ddx5/Ddx17 in cell proliferation. The U3snoRNA is shown here to be essential for the proliferation and viability of human cells. Moreover, it was found that U3snoRNA interacts with Argonaute 2 in the RNA-induced silencing complexes (RISC), pointing to a microRNA-like function. For this reason, the 3′ untranslated region of the A-kinase anchor protein 9 (AKAP9)-mRNA could be identified as a potential target.

Clustering and Functional Coupling of Diverse Ion Channels and Signaling Proteins Revealed by Super-resolution STORM Microscopy in Neurons

The fidelity of neuronal signaling requires organization of signaling molecules into macromolecular complexes, whose components are in intimate proximity. The intrinsic diffraction limit of light makes visualization of individual signaling complexes using visible light extremely difficult. However, using super-resolution stochastic optical reconstruction microscopy (STORM), we observed intimate association of individual molecules within signaling complexes containing ion channels (M-type K+, L-type Ca2+, or TRPV1 channels) and G protein-coupled receptors coupled by the scaffolding protein A-kinase-anchoring protein (AKAP)79/150. Some channels assembled as multi-channel supercomplexes. Surprisingly, we identified novel layers of interplay within macromolecular complexes containing diverse channel types at the single-complex level in sensory neurons, dependent on AKAP79/150. Electrophysiological studies revealed that such ion channels are functionally coupled as well. Our findings illustrate the novel role of AKAP79/150 as a molecular coupler of different channels that conveys crosstalk between channel activities within single microdomains in tuning the physiological response of neurons.

Sperm with fibrous sheath dysplasia and anomalies in head-neck junction: focus on centriole and centrin 1

Spermatozoa with a rare combination of two monomorphic sperm defects, dysplasia of the fibrous sheath (DFS) and alterations in head-mid-piece junction were analysed. The main focus was to explore the status of the centriole, a key organisation during fertilisation, using the centrin 1, a calcium-binding protein linked to this structure. The sperm quality was examined by light, scanning and transmission electron microscopy (SEM, TEM); immunocytochemistry was performed for tubulin, A-kinase anchor protein 4 (AKAP4) and centrin 1. Spermatozoa showed DFS defect associated with anomalies in head-tail attachment detected by SEM and TEM. Immunolocalisation of tubulin, AKAP4 and centrin 1 confirmed these alterations. Centrin 1 was visible in 67% of spermatozoa (in only 13% centrin localised in a normal position); in the majority of sperm centrin 1's location was altered, sometimes bent; often four spots, indicating the presence of two implantation fossae, were detected. At the centriolar level, immunoreactive fragments, frequently invading the entire short and thick tail, were observed. Centrin 1 is an essential component of the spermatozoa connecting piece and plays a role in centrosome dynamics during sperm morphogenesis and in zygotes and early embryos during spindle assembly. It is important to shed light on these rare conditions in order to better manage the patients during assisted reproductive technology.

The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β

The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.

Malonate in the nucleotide-binding site traps human AKAP18γ/δ in a novel conformational state.

A-kinase anchoring proteins (AKAPs) are a family of proteins that provide spatiotemporal resolution of protein kinase A (PKA) phosphorylation. In the myocardium, PKA and AKAP18γ/δ are found in complex with sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and phospholamban (PLB). This macromolecular complex provides a means by which anchored PKA can dynamically regulate cytoplasmic Ca(2+) release and re-uptake. For this reason, AKAP18γ/δ presents an interesting drug target with therapeutic potential in cardiovascular disease. The crystal structure of the central domain of human AKAP18γ has been determined at the atomic resolution of 1.25 Å. This first structure of human AKAP18γ is trapped in a novel conformation by a malonate molecule bridging the important R-loop with the 2H phosphoesterase motif. Although the physiological substrate of AKAP18γ is currently unknown, a potential proton wire deep in the central binding crevice has been indentified, leading to bulk solvent below the R-loop. Malonate complexed with AKAP18γ at atomic resolution provides an excellent starting point for structure-guided drug design.