AKAP350 enables p150glued /EB1 interaction at the spindle poles.

A-kinase anchoring protein 350 (AKAP350) is a centrosomal/Golgi scaffold protein, critical for the regulation of microtubule dynamics. AKAP350 recruits end-binding protein 1 (EB1) to the centrosome in mitotic cells, ensuring proper spindle orientation in epithelial cells. AKAP350 also interacts with p150glued, the main component of the dynactin complex. In the present work, we found that AKAP350 localized p150glued to the spindle poles, facilitating p150glued/EB1 interaction at these structures. Our results further showed that the decrease in AKAP350 expression reduced p150glued localization at astral microtubules and impaired the elongation of astral microtubules during anaphase. Overall, this study provides mechanistic data on how microtubule regulatory proteins gather to define microtubule dynamics in mitotic cells.

Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis.

It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus. Mutations of key residues to different amino acids perturb AKAP95 condensation in opposite directions. Importantly, the activity of AKAP95 in splice regulation is abolished by disruption of condensation, significantly impaired by hardening of condensates, and regained by substituting its condensation-mediating region with other condensation-mediating regions from irrelevant proteins. Moreover, the abilities of AKAP95 in regulating gene expression and supporting tumorigenesis require AKAP95 to form condensates with proper liquidity and dynamicity. These results link phase separation to tumorigenesis and uncover an important role of appropriate biophysical properties of protein condensates in gene regulation and cancer.

mAKAPß signalosomes - A nodal regulator of gene transcription associated with pathological cardiac remodeling.

Striated myocytes compose about half of the cells of the heart, while contributing the majority of the heart's mass and volume. In response to increased demands for pumping power, including in diseases of pressure and volume overload, the contractile myocytes undergo non-mitotic growth, resulting in increased heart mass, i.e. cardiac hypertrophy. Myocyte hypertrophy is induced by a change in the gene expression program driven by the altered activity of transcription factors and co-repressor and co-activator chromatin-associated proteins. These gene regulatory proteins are subject to diverse post-translational modifications and serve as nuclear effectors for intracellular signal transduction pathways, including those controlled by cyclic nucleotides and calcium ion. Scaffold proteins contribute to the underlying architecture of intracellular signaling networks by targeting signaling enzymes to discrete intracellular compartments, providing specificity to the regulation of downstream effectors, including those regulating gene expression. Muscle A-kinase anchoring protein ß (mAKAPß) is a well-characterized scaffold protein that contributes to the regulation of pathological cardiac hypertrophy. In this review, we discuss the mechanisms how this prototypical scaffold protein organizes signalosomes responsible for the regulation of class IIa histone deacetylases and cardiac transcription factors such as NFAT, MEF2, and HIF-1α, as well as how this signalosome represents a novel therapeutic target for the prevention or treatment of heart failure.

Induction of ß-adrenergic metaplasticity of LTP requires intact anchoring of PKA.

Beta-adrenergic receptors (ß-ARs) prime hippocampal synapses to stabilize long-term potentiation (LTP). This "metaplasticity" can persist for 1-2 h after pharmacologic activation of ß-ARs. It requires activation of PKA (cAMP-dependent protein kinase) during ß-AR priming. A-kinase anchoring proteins (AKAPs) tether PKA to downstream signaling proteins. We hypothesized that induction of this metaplasticity requires intact functioning of AKAPs. Acute application of stearated ht31, a membrane-permeant inhibitor of AKAPs, either during ß-AR activation 30 min prior to LTP induction or during LTP induction, attenuated the persistence of LTP. A control, inactive ht31 peptide did not affect ß-AR-mediated metaplasticity. These findings implicate PKA anchoring in the induction of ß-adrenergic metaplasticity of LTP.

Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca2+-Permeable AMPA Receptors.

Neuronal information processing requires multiple forms of synaptic plasticity mediated by NMDARs and AMPA-type glutamate receptors (AMPARs). These plasticity mechanisms include long-term potentiation (LTP) and long-term depression (LTD), which are Hebbian, homosynaptic mechanisms locally regulating synaptic strength of specific inputs, and homeostatic synaptic scaling, which is a heterosynaptic mechanism globally regulating synaptic strength across all inputs. In many cases, LTP and homeostatic scaling regulate AMPAR subunit composition to increase synaptic strength via incorporation of Ca2+-permeable receptors (CP-AMPAR) containing GluA1, but lacking GluA2, subunits. Previous work by our group and others demonstrated that anchoring of the kinase PKA and the phosphatase calcineurin (CaN) to A-kinase anchoring protein (AKAP) 150 play opposing roles in regulation of GluA1 Ser845 phosphorylation and CP-AMPAR synaptic incorporation during hippocampal LTP and LTD. Here, using both male and female knock-in mice that are deficient in PKA or CaN anchoring, we show that AKAP150-anchored PKA and CaN also play novel roles in controlling CP-AMPAR synaptic incorporation during homeostatic plasticity in hippocampal neurons. We found that genetic disruption of AKAP-PKA anchoring prevented increases in Ser845 phosphorylation and CP-AMPAR synaptic recruitment during rapid homeostatic synaptic scaling-up induced by combined blockade of action potential firing and NMDAR activity. In contrast, genetic disruption of AKAP-CaN anchoring resulted in basal increases in Ser845 phosphorylation and CP-AMPAR synaptic activity that blocked subsequent scaling-up by preventing additional CP-AMPAR recruitment. Thus, the balanced, opposing phospho-regulation provided by AKAP-anchored PKA and CaN is essential for control of both Hebbian and homeostatic plasticity mechanisms that require CP-AMPARs.SIGNIFICANCE STATEMENT Neuronal circuit function is shaped by multiple forms of activity-dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts strength of individual synapses and homeostatic plasticity that adjusts overall strength of all synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR regulation by the kinase PKA and phosphatase CaN coanchored to the scaffold protein AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic strength during homeostatic plasticity. These novel findings significantly expand our understanding of homeostatic plasticity mechanisms and further emphasize how intertwined they are with LTP and LTD.

Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.

Critically ill patients are commonly treated with high levels of oxygen, hyperoxia, for prolonged periods of time. Unfortunately, extended exposure to hyperoxia can exacerbate respiratory failure and lead to a high mortality rate. Mitochondrial A-kinase anchoring protein (Akap) has been shown to regulate mitochondrial function. It has been reported that, under hypoxic conditions, Akap121 undergoes proteolytic degradation and promotes cardiac injury. However, the role of Akap1 in hyperoxia-induced acute lung injury (ALI) is largely unknown. To address this gap in our understanding of Akap1, we exposed wild-type ( wt) and Akap1-/- mice to 100% oxygen for 48 h, a time point associated with lung damage in the murine model of ALI. We found that under hyperoxia, Akap1-/- mice display increased levels of proinflammatory cytokines, immune cell infiltration, and protein leakage in lungs, as well as increased alveolar capillary permeability compared with wt controls. Further analysis revealed that Akap1 deletion enhances lung NF-κB p65 activity as assessed by immunoblotting and DNA-binding assay and mitochondrial autophagy-related markers, PINK1 and Parkin. Ultrastructural analysis using electron microscopy revealed that Akap1 deletion was associated with remarkably aberrant mitochondria and lamellar bodies in type II alveolar epithelial cells. Taken together, these results demonstrate that Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.

AKAP200 promotes Notch stability by protecting it from Cbl/lysosome-mediated degradation in Drosophila melanogaster.

AKAP200 is a Drosophila melanogaster member of the "A Kinase Associated Protein" family of scaffolding proteins, known for their role in the spatial and temporal regulation of Protein Kinase A (PKA) in multiple signaling contexts. Here, we demonstrate an unexpected function of AKAP200 in promoting Notch protein stability. In Drosophila, AKAP200 loss-of-function (LOF) mutants show phenotypes that resemble Notch LOF defects, including eye patterning and sensory organ specification defects. Through genetic interactions, we demonstrate that AKAP200 interacts positively with Notch in both the eye and the thorax. We further show that AKAP200 is part of a physical complex with Notch. Biochemical studies reveal that AKAP200 stabilizes endogenous Notch protein, and that it limits ubiquitination of Notch. Specifically, our genetic and biochemical evidence indicates that AKAP200 protects Notch from the E3-ubiquitin ligase Cbl, which targets Notch to the lysosomal pathway. Indeed, we demonstrate that the effect of AKAP200 on Notch levels depends on the lysosome. Interestingly, this function of AKAP200 is fully independent of its role in PKA signaling and independent of its ability to bind PKA. Taken together, our data indicate that AKAP200 is a novel tissue specific posttranslational regulator of Notch, maintaining high Notch protein levels and thus promoting Notch signaling.

AKAP150 involved in paclitaxel-induced neuropathic pain via inhibiting CN/NFAT2 pathway and downregulating IL-4.

Antitubulin chemotherapeutics agents, such as paclitaxel, are effective chemotherapy drugs for cancer treatment. However, painful neuropathy is a major adverse effect limiting the wider application of chemotherapeutics. In this study, we found that A-kinase anchor protein 150 (AKAP150) was significantly upregulated after paclitaxel injection. Inhibition of AKAP150 via siRNA or AKAP150flox/flox in rodents alleviated the pain behavior induced by paclitaxel, and partly restored the decreased calcineurin (CN) phosphatase activity after paclitaxel treatment. Paclitaxel decreased the expression of anti-inflammatory cytokine interleukin-4 (IL-4), and intrathecal injections of IL-4 effectively alleviated paclitaxel-induced hypersensitivity and the frequency of dorsal root ganglion (DRG) neurons action potential. The decreased CN enzyme activity, resulted in reduced protein expression of nuclear factor of activated T cells 2 (NFAT2) in cell nuclei. Chromatin immunoprecipitation showed that, NFAT2 binds to the IL-4 gene promoter regulating the protein expression of IL-4. Overexpression of NFAT2 by intrathecal injection of the AAV5-NFAT2-GFP virus alleviated the pain behavior induced by paclitaxel via increasing the expression of IL-4. Knocked down AKAP150 by siRNA or AAV5-Cre-GFP partly restored the expression of IL-4 in DRG. Our results indicated that regulation of IL-4 via the CN/NFAT2 pathway mediated by AKAP150 could be a pivotal treatment target for paclitaxel-induced neuropathic pain and or other neuropsychiatric disorders.

AKAP95-mediated nuclear anchoring of PKA mediates cortisol-induced PTGS2 expression in human amnion fibroblasts.

Phosphorylation of the transcription factors cyclic adenosine monophosphate response element-binding protein (CREB) and signal transducer and activator of transcription 3 (STAT3) by protein kinase A (PKA) is required for the cortisol-induced production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in human amnion fibroblasts, which critically mediates human parturition (labor). We found that PKA was confined in the nucleus by A-kinase-anchoring protein 95 (AKAP95) in amnion fibroblasts and that this localization was key to the cortisol-induced expression of PTGS2, the gene encoding COX-2. Cortisol increased the abundance of nuclear PKA by stimulating the expression of the gene encoding AKAP95. Knockdown of AKAP95 not only reduced the amounts of nuclear PKA and phosphorylated CREB but also attenuated the induction of PTGS2 expression in primary human amnion fibroblasts treated with cortisol, whereas the phosphorylation of STAT3 in response to cortisol was not affected. The abundances of AKAP95, phosphorylated CREB, and COX-2 were markedly increased in human amnion tissue after labor compared to those in amnion tissues from cesarean sections without labor. These results highlight an essential role for PKA that is anchored in the nucleus by AKAP95 in the phosphorylation of CREB and the consequent induction of COX-2 expression by cortisol in amnion fibroblasts, which may be important in human parturition.

AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair.

Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12-ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5' strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12-ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

Pivotal role of AKAP121 in mitochondrial physiology.

In this Perspective, we discuss some recent developments in the study of the mitochondrial scaffolding protein AKAP121 (also known as AKAP1, or AKAP149 as the human homolog), with an emphasis on its role in mitochondrial physiology. AKAP121 has been identified to function as a key regulatory molecule in several mitochondrial events including oxidative phosphorylation, the control of membrane potential, fission-induced apoptosis, maintenance of mitochondrial Ca(2+)homeostasis, and the phosphorylation of various mitochondrial respiratory chain substrate molecules. Furthermore, we discuss the role of hypoxia in prompting cellular stress and damage, which has been demonstrated to mediate the proteosomal degradation of AKAP121, leading to an increase in reactive oxgyen species production, mitochondrial dysfunction, and ultimately cell death.

A-kinase anchoring protein 2 is required for calcitonin-mediated invasion of cancer cells.

Expression of neuropeptide calcitonin (CT) and its receptor (CTR) is frequently elevated in prostate cancers (PCs) and activation of CT-CTR axis in non-invasive PC cells induces an invasive phenotype. Specific, cell-permeable inhibitors of protein kinase A abolish CTR-stimulated invasion of PC cells. Since PKA is ubiquitously distributed in cells, the present study examined the mechanism(s) by which CTR-stimulated PKA activity is regulated in time and space. CT reduced cell adhesion but increased invasion of PC cells. Both these actions were abolished by st-Ht31 inhibitory peptide suggesting the involvement of an A-kinase anchoring protein (AKAP) in CT action. Next, we identified the AKAP associated with CT action by the subtraction of potential AKAP candidates using siRNAs. Knock-down of membrane-associated AKAP2, but not other AKAPs, abolished CT-stimulated invasion. Stable knock-down of AKAP2 in PC3-CTR cells remarkably decreased their cell proliferation, invasion, clonogenicity and ability to form orthotopic tumors and distant metastases in nude mice. Re-expression of AKAP2-wt restored these characteristics. Primary PC specimens displayed remarkable upregulation of CTR/AKAP2 expression as compared to benign prostates. Metastatic cancers displayed significantly higher CTR/AKAP2 expression than localized cancers. These results for the first time demonstrate that AKAP2 is expressed in human prostates, its expression is elevated in metastatic prostate cancer, and the knock-down of its expression remarkably decreased tumorigenicity and metastatic ability of prostate cancer cells. AKAP2 may serve as a critical component of CTR-mediated oncogenic actions.

M-current preservation contributes to anticonvulsant effects of valproic acid.

Valproic acid (VPA) has been widely used for decades to treat epilepsy; however, its mechanism of action remains poorly understood. Here, we report that the anticonvulsant effects of nonacute VPA treatment involve preservation of the M-current, a low-threshold noninactivating potassium current, during seizures. In a wide variety of neurons, activation of Gq-coupled receptors, such as the m1 muscarinic acetylcholine receptor, suppresses the M-current and induces hyperexcitability. We demonstrated that VPA treatment disrupts muscarinic suppression of the M-current and prevents resultant agonist-induced neuronal hyperexcitability. We also determined that VPA treatment interferes with M-channel signaling by inhibiting palmitoylation of a signaling scaffold protein, AKAP79/150, in cultured neurons. In a kainate-induced murine seizure model, administration of a dose of an M-channel inhibitor that did not affect kainate-induced seizure transiently eliminated the anticonvulsant effects of VPA. Retigabine, an M-channel opener that does not open receptor-suppressed M-channels, provided anticonvulsant effects only when administered prior to seizure induction in control animals. In contrast, treatment of VPA-treated mice with retigabine induced anticonvulsant effects even when administered after seizure induction. Together, these results suggest that receptor-induced M-current suppression plays a role in the pathophysiology of seizures and that preservation of the M-current during seizures has potential as an effective therapeutic strategy.

Pharmacological targeting of AKAP-directed compartmentalized cAMP signalling.

The second messenger cyclic adenosine monophosphate (cAMP) can bind and activate protein kinase A (PKA). The cAMP/PKA system is ubiquitous and involved in a wide array of biological processes and therefore requires tight spatial and temporal regulation. Important components of the safeguard system are the A-kinase anchoring proteins (AKAPs), a heterogeneous family of scaffolding proteins defined by its ability to directly bind PKA. AKAPs tether PKA to specific subcellular compartments, and they bind further interaction partners to create local signalling hubs. The recent discovery of new AKAPs and advances in the field that shed light on the relevance of these hubs for human disease highlight unique opportunities for pharmacological modulation. This review exemplifies how interference with signalling, particularly cAMP signalling, at such hubs can reshape signalling responses and discusses how this could lead to novel pharmacological concepts for the treatment of disease with an unmet medical need such as cardiovascular disease and cancer.

Targeting of type I protein kinase A to lipid rafts is required for platelet inhibition by the 3',5'-cyclic adenosine monophosphate-signaling pathway.

BACKGROUND: Platelet adhesion to von Willebrand factor (VWF) is modulated by 3',5'-cyclic adenosine monophosphate (cAMP) signaling through protein kinase A (PKA)-mediated phosphorylation of glycoprotein (GP)Ibß. A-kinase anchoring proteins (AKAPs) are proposed to control the localization and substrate specificity of individual PKA isoforms. However, the role of PKA isoforms in regulating the phosphorylation of GPIbß and platelet response to VWF is unknown. OBJECTIVES: We wished to determine the role of PKA isoforms in the phosphorylation of GPIbß and platelet activation by VWF as a model for exploring the selective partitioning of cAMP signaling in platelets. RESULTS: The two isoforms of PKA in platelets, type I (PKA-I) and type II (PKA-II), were differentially localized, with a small pool of PKA-I found in lipid rafts. Using a combination of Far Western blotting, immunoprecipitation, proximity ligation assay and cAMP pull-down we identified moesin as an AKAP that potentially localizes PKA-I to rafts. Introduction of cell-permeable anchoring disruptor peptide, RI anchoring disruptor (RIAD-Arg11 ), to block PKA-I/AKAP interactions, uncoupled PKA-RI from moesin, displaced PKA-RI from rafts and reduced kinase activity in rafts. Examination of GPIbß demonstrated that it was phosphorylated in response to low concentrations of PGI2 in a PKA-dependent manner and occurred primarily in lipid raft fractions. RIAD-Arg11 caused a significant reduction in raft-localized phosphoGPIbß and diminished the ability of PGI2 to regulate VWF-mediated aggregation and thrombus formation in vitro. CONCLUSION: We propose that PKA-I-specific AKAPs in platelets, including moesin, organize a selective localization of PKA-I required for phosphorylation of GPIbß and contribute to inhibition of platelet VWF interactions.

Histone Deacetylase Inhibition Rescues Maternal Deprivation-Induced GABAergic Metaplasticity through Restoration of AKAP Signaling.

Adverse early-life experiences such as child neglect and abuse increase the risk of developing addiction and stress-related disorders through alterations in motivational systems including the mesolimbic dopamine (DA) pathway. Here we investigated whether a severe early-life stress (i.e., maternal deprivation, MD) promotes DA dysregulation through an epigenetic impairment of synaptic plasticity within ventral tegmental area (VTA) DA neurons. Using a single 24-hr episode of MD and whole-cell patch clamp recording in rat midbrain slices, we show that MD selectively induces long-term depression (LTD) and shifts spike timing-dependent plasticity (STDP) toward LTD at GABAergic synapses onto VTA DA neurons through epigenetic modifications of postsynaptic scaffolding A-kinase anchoring protein 79/150 (AKAP79/150) signaling. Histone deacetylase (HDAC) inhibition rescues GABAergic metaplasticity and normalizes AKAP signaling in MD animals. MD-induced reversible HDAC-mediated GABAergic dysfunction within the VTA may be a mechanistic link for increased propensity to mental health disorders following MD.

AKAP5 signaling complexes: focal points and functional properties.

OBJECTIVE: Kinase Anchoring Proteins (AKAPs) have evolved to regulate the spatial and temporal organization of cellular signal transduction. As a typical member, AKAP5 which consisting of three orthologues: bovine AKAP75, rodent AKAP150 and human AKAP79, is the best known model in the anchoring and targeting properties. It is shown that AKAP5 can bind ß2-adrenergic receptor, which is a member of GPCR superfamily, and orchestrate the interactions of various protein kinases, protein phosphatases and cytoskeletal element. AKAP5 is originally identified as a component of the postsynaptic density in neurons and plays a vital role in modulating neuronal activities. Subsequently, the AKAP5 complexes are also detected in other tissues and participated in various processes.