Sensitization of glutamate receptor-mediated pain behaviour via nerve growth factor-dependent phosphorylation of transient receptor potential V1 under inflammatory conditions.

BACKGROUND AND PURPOSE: Glutamate and metabotropic glutamate (mGlu) receptors on primary sensory neurons are crucial in modulating pain sensitivity. However, it is unclear how inflammation affects mGlu receptor-mediated nociceptive responses. We therefore investigated the effects of mGlu1/5 receptor agonists on pain-related behaviour during persistent inflammation and their underlying mechanisms. EXPERIMENTAL APPROACH: Effects of a mGlu1/5 receptor agonist on pain-related behaviour during inflammation was assessed in mice. Intracellular calcium responses, membrane current responses, and protein expression in primary sensory neurons were examined using cultured dorsal root ganglion (DRG) neurons, dissociated from wild-type and gene knockout mice. KEY RESULTS: Persistent inflammation induced by complete Freund's adjuvant increased the duration of mGlu1/5 receptor-mediated pain behaviour, which was antagonized by inhibition of nerve growth factor (NGF)-tropomyosin receptor kinase A (TrkA) signalling. Calcium imaging revealed that NGF treatment increased the number of cultured DRG neurons responding to mGlu1/5 receptor activation. Stimulation of mGlu1/5 receptors in NGF-treated DRG neurons induced inward currents through TRPV1 channels in association with PLC but not with IP3 receptors. NGF treatment also increased the number of neurons responding to a DAG analogue via TRPV1 channel activation. Furthermore, NGF up-regulated expression of TRPV1 and A-kinase anchoring protein 5 (AKAP5), resulting in increased AKAP5-dependent TRPV1 phosphorylation. AKAP5 knockout mice did not exhibit mGlu1/5 receptor-mediated excitation in NGF-treated DRG neurons or pain response facilitation under inflammatory conditions. CONCLUSIONS AND IMPLICATIONS: NGF augments glutamate- and mGlu1/5 receptor-mediated excitation of nociceptive neurons by AKAP5-dependent phosphorylation of TRPV1 channels, potentiating hypersensitivity to glutamate in inflamed tissues.

Maturation of thalamocortical synapses in the somatosensory cortex depends on neocortical AKAP5 expression.

Sensory cortex topographic maps consist of organized arrays of thalamocortical afferents (TCAs) that project into distinct areas of the cortex. Formation of topographic maps in sensory cortices is a prerequisite for functional maturation of the neocortex. Studies have shown that the formation of topographic maps and the maturation of thalamocortical synapses in the somatosensory cortex depend on the cyclic adenosine 5'-monophosphate-(cAMP)-protein kinase A (PKA) signaling pathway. AKAP5 is a scaffold protein (also called AKAP79 in humans or AKAP150 in rodents; AKAP79/150) that serves as a signaling hub that links cAMP and PKA signaling. Whether AKAP5 plays a role in topographic map formation and the maturation of thalamocortical synapses during development of the somatosensory cortex is still unknown. Here, we generated cortex-specific AKAP5-knockout mice (CxAKAP5KO) to examine its roles in somatosensory cortex development. We found that CxAKAP5KO mice displayed impaired cortical barrel maps. Electrophysiological recordings showed that the AMPA/NMDA ratio was reduced, and silent synapses were increased in thalamocortical synapses of CxAKAP5KO mice during postnatal development. Morphological analysis of layer IV cortical neurons demonstrated that dendritic refinement of these neurons was abnormal. These results indicate that AKAP5 is necessary for both topographic map formation and maturation of thalamocortical synapses as well as morphological development of cortical neurons in the somatosensory cortex.

Human OVGP1 enhances tyrosine phosphorylation of proteins in the fibrous sheath involving AKAP3 and increases sperm-zona binding.

PURPOSE: To investigate if the recombinant human oviduct-specific glycoprotein (rHuOVGP1)-enhanced tyrosine-phosphorylated (pY) proteins are components of specific structure(s) of the sperm tail and if rHuOVGP1 binds to the oocyte and enhances sperm-egg binding. METHODS: Immunofluorescent staining and confocal microscopy were performed to examine the localization of pY proteins, outer dense fiber (ODF), and A-Kinase Associated Protein 3 (AKAP3) in human sperm during capacitation. Western blot and immunoprecipitation were employed to analyze protein levels of pY proteins and AKAP3. Immunofluorescent staining was performed to examine the binding of rHuOVGP1 to human oocytes. The effect of rHuOVGP1 on enhancing sperm-zona binding was examined using hemizona assay. RESULTS: pY proteins were detected mainly in the fibrous sheath (FS) surrounding the ODF with a relatively weak immunoreaction in the neck and mid-piece. Western blot analysis revealed co-migration of the pY 105 kDa protein with AKAP3, which was further confirmed by immunoprecipitation correlating immunofluorescent results of co-localization of pY proteins with AKAP3 in the sperm tail. rHuOVGP1 binds specifically to the zona pellucida (ZP) of human oocytes. Prior incubation of sperm and/or ZP with rHuOVGP1 increased sperm-egg binding. CONCLUSIONS: The present study revealed that one of the major rHuOVGP1-enhanced pY proteins could be AKAP3 of the FS and that rHuOVGP1 is capable of binding to human ZP and its presence in the medium results in an increase in sperm-zona binding. Supplement of rHuOVGP1 in in vitro fertilization media could be beneficial for enhancement of the fertilizing ability of human sperm.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways.

Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin.

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca(2+) -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca(2+) -dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca(2+) -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.

PKA/AKAP1 and PP2A/Bß2 regulate neuronal morphogenesis via Drp1 phosphorylation and mitochondrial bioenergetics.

Mitochondrial shape is determined by fission and fusion reactions, perturbation of which can contribute to neuronal injury and disease. Mitochondrial fission is catalyzed by dynamin-related protein 1 (Drp1), a large GTPase of the dynamin family that is highly expressed in neurons and regulated by various posttranslational modifications, including phosphorylation. We report here that reversible phosphorylation of Drp1 at a conserved Ser residue by an outer mitochondrial kinase (PKA/AKAP1) and phosphatase (PP2A/Bß2) impacts dendrite and synapse development in cultured rat hippocampal neurons. PKA/AKAP1-mediated phosphorylation of Drp1 at Ser656 increased mitochondrial length and dendrite occupancy, enhancing dendritic outgrowth but paradoxically decreasing synapse number and density. Opposing PKA/AKAP1, PP2A/Bß2-mediated dephosphorylation of Drp1 at Ser656 fragmented and depolarized mitochondria and depleted them from dendrites, stunting dendritic outgrowth but augmenting synapse formation. Raising and lowering intracellular calcium reproduced the effects of dephospho-Drp1 and phospho-Drp1on dendrite and synapse development, respectively, while boosting mitochondrial membrane potential with l-carnitine-fostered dendrite at the expense of synapse formation without altering mitochondrial size or distribution. Thus, outer mitochondrial PKA and PP2A regulate neuronal development by inhibiting and promoting mitochondrial division, respectively. We propose that the bioenergetic state of mitochondria, rather than their localization or shape per se, is the key effector of Drp1, altering calcium homeostasis to modulate neuronal morphology and connectivity.

Functional roles of a C-terminal signaling complex of CaV1 channels and A-kinase anchoring protein 15 in brain neurons.

Regulation of CaV1.2 channels in cardiac myocytes by the ß-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ß-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2ΔDCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2ΔDCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.

Ca2+/calmodulin-dependent protein kinase II inhibitors disrupt AKAP79-dependent PKC signaling to GluA1 AMPA receptors.

GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31-52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31-52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31-52) in the absence (apoCaM) but not the presence of Ca(2+). In contrast, apoCaM association to AKAP79(31-52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31-52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII.

AKAP79 selectively enhances protein kinase C regulation of GluR1 at a Ca2+-calmodulin-dependent protein kinase II/protein kinase C site.

Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents approximately 20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses.

DNA copy-number analysis in bipolar disorder and schizophrenia reveals aberrations in genes involved in glutamate signaling.

Using bacterial artificial chromosome (BAC) array comparative genome hybridization (aCGH) at approximately 1.4 Mbp resolution, we screened post-mortem brain DNA from bipolar disorder cases, schizophrenia cases and control individuals (n=35 each) for DNA copy-number aberrations. DNA copy number is a largely unexplored source of human genetic variation that may contribute risk for complex disease. We report aberrations at four loci which were seen in affected but not control individuals, and which were verified by quantitative real-time PCR. These aberrant loci contained the genes encoding EFNA5, GLUR7, CACNG2 and AKAP5; all brain-expressed proteins with known or postulated roles in neuronal function, and three of which (GLUR7, CACNG2 and AKAP5) are involved in glutamate signaling. A second cohort of psychiatric samples was also tested by quantitative PCR using the primer/probe sets for EFNA5, GLUR7, CACNG2 and AKAP5, and samples with aberrant copy number were found at three of the four loci (GLUR7, CACNG2 and AKAP5). Further scrutiny of these regions may reveal insights into the etiology and genetic risk factors for these complex psychiatric disorders.

Autonomic control of cardiac action potentials: role of potassium channel kinetics in response to sympathetic stimulation.

I(Ks), the slowly activating component of the delayed rectifier current, plays a major role in repolarization of the cardiac action potential (AP). Genetic mutations in the alpha- (KCNQ1) and beta- (KCNE1) subunits of I(Ks) underlie Long QT Syndrome type 1 and 5 (LQT-1 and LQT-5), respectively, and predispose carriers to the development of polymorphic ventricular arrhythmias and sudden cardiac death. beta-adrenergic stimulation increases I(Ks) and results in rate dependent AP shortening, a control system that can be disrupted by some mutations linked to LQT-1 and LQT-5. The mechanisms by which I(Ks) regulates action potential duration (APD) during beta-adrenergic stimulation at different heart rates are not known, nor are the consequences of mutation induced disruption of this regulation. Here we develop a complementary experimental and theoretical approach to address these questions. We reconstituted I(Ks) in CHO cells (ie, KCNQ1 coexpressed with KCNE1 and the adaptator protein Yotiao) and quantitatively examined the effects of beta-adrenergic stimulation on channel kinetics. We then developed theoretical models of I(Ks) in the absence and presence of beta-adrenergic stimulation. We simulated the effects of sympathetic stimulation on channel activation (speeding) and deactivation (slowing) kinetics on the whole cell action potential under different pacing conditions. The model suggests these kinetic effects are critically important in rate-dependent control of action potential duration. We also investigate the effects of two LQT-5 mutations that alter kinetics and impair sympathetic stimulation of I(Ks) and show the likely mechanism by which they lead to tachyarrhythmias and indicate a distinct role of I(KS) kinetics in this electrical dysfunction. The full text of this article is available online at http://circres.ahajournals.org.

Nesprin-1alpha contributes to the targeting of mAKAP to the cardiac myocyte nuclear envelope.

Muscle A-kinase anchoring protein (mAKAP) is a scaffold protein found principally at the nuclear envelope of striated myocytes. mAKAP maintains a complex consisting of multiple signal transduction molecules including the cAMP-dependent protein kinase A, the ryanodine receptor calcium release channel, phosphodiesterase type 4D3, and protein phosphatase 2A. By an unknown mechanism, a domain containing spectrin repeats is responsible for targeting mAKAP to the nuclear envelope. We now demonstrate that the integral membrane protein nesprin-1alpha serves as a receptor for mAKAP on the nuclear envelope in cardiac myocytes. Nesprin-1alpha is inserted into the nuclear envelope by a conserved, C-terminal, klarsicht-related transmembrane domain and forms homodimers by the binding of an amino-terminal spectrin repeat domain. Through the direct binding of the nesprin-1alpha amino-terminal dimerization domain to the third mAKAP spectrin repeat, nesprin-1alpha targets mAKAP to the nuclear envelope. In turn, overexpression of these spectrin repeat domains in myocytes can displace mAKAP from nesprin-1alpha.

AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular channel (CLIC) family member.

AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611-6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178-410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.

mAKAP and the ryanodine receptor are part of a multi-component signaling complex on the cardiomyocyte nuclear envelope.

The physical association of regulatory enzymes and ion channels at relevant intracellular sites contributes to the diversity and specificity of second messenger-mediated signal transduction in cells. mAKAP is a scaffolding protein that targets the cAMP-dependent protein kinase A and phosphodiesterase type 4D3 to the nuclear envelope of differentiated cardiac myocytes. Here we present data that the mAKAP signaling complex also includes nuclear envelope-resident ryanodine receptors and protein phosphatase 2A. The ryanodine receptor is the major cardiac ion channel responsible for calcium-induced calcium release from intracellular calcium ion stores. As demonstrated by a combination of immunohistochemistry and tissue fractionation, mAKAP is targeted specifically to the nuclear envelope, whereas the ryanodine receptor is present at both the sarcoplasmic reticulum and nuclear envelope intracellular membrane compartments. At the nuclear envelope, a subset of cardiac ryanodine receptor is bound to mAKAP and via the association with mAKAP may be regulated by protein kinase A-mediated phosphorylation. By binding protein kinase A and ryanodine receptor, mAKAP may serve as the scaffold for a cAMP- and calcium ion-sensitive signaling complex.

Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13.

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity. Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation. Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13). PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD. Co-expression of C. botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor. Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation. Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.

Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene.

A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI approximately 3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, DeltaDAKAP200. DeltaDAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. DeltaDAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and DeltaDAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; DeltaDAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.

Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.

A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin.

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

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.