Expression Of A-kinase Anchoring Protein Research Articles

Decreased AKAP4/PKA signaling pathway in high DFI sperm affects sperm capacitation.

The sperm DNA fragmentation index (DFI) is a metric used to assess DNA fragmentation within sperm. During in vitro fertilization-embryo transfer (IVF-ET), high sperm DFI can lead to a low fertilization rate, poor embryo development, early miscarriage, etc. A kinase anchoring protein (AKAP) is a scaffold protein that can bind protein kinase A (PKA) to subcellular sites of specific substrates and protects the biophosphorylation reaction. Sperm protein antigen 17 (SPA17) can also bind to AKAP. This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI (H-DFI) patients during IVF-ET. In addition, the study investigates the expression of AKAP, protein kinase A regulatory subunit (PKARII), and SPA17 between H-DFI and low sperm DFI (L-DFI) patients. SPA17 at the transcriptional level is abnormal, the translational level increases in H-DFI patients, and the expression of AKAP4/PKARII protein decreases. H2O2 has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI. It indicates that H2O2 increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARII protein expression. These processes inhibit sperm capacitation and reduce acrosomal reactions. Embryo culture data and IVF outcomes have been documented. The H-DFI group has a lower fertilization rate. Therefore, the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARII proteins, blocked sperm capacitation, and reduced occurrence of acrosome reaction.

A-Kinase anchor protein 4 (AKAP4) may be considered as a potential early diagnostic breast cancer marker detectable in blood

PurposeBreast cancer is the most common type of cancer-related death in women worldwide. Early diagnosis via cancer screening biomarkers has a major impact on improving clinical outcomes and overall survival leads to better cancer management. Herein, we examined the expression of A-Kinase Anchor Protein 4 (AKAP4) as a Cancer Testis Antigen (CTA) in breast cancer tumors and traced it in the peripheral blood as a possible diagnostic breast cancer marker. Material and methodsTo evaluate the expression of AKAP4 gene nested real-time RT-PCR method with SYBR green dye were used in the peripheral blood and breast tissues of 80 breast cancer patients in comparison with normal controls. ResultsThe data revealed that the existence of AKAP4 gene expression in all the stages of breast cancer in tumor tissues and its subsequent detection in peripheral blood could be regarded as a possible biomarker for breast cancer diagnosis even at the early stages. The AKAP4 expression was detected somewhat in the normal margin of the tumor compared with normal breast tissue with no AKAP4 expression which indicates the bystander effect. The peripheral blood AKAP4 expression was significantly higher in stage IV, nodal involvement, and distant metastasis. ConclusionAKAP4 gene expression analysis in blood could be regarded as a possible early diagnostic biomarker in breast cancer that is an indispensable requirement for better management of cancer.

Low expression of A-kinase anchor protein 5 predicts poor prognosis in non-mucin producing stomach adenocarcinoma based on TCGA data.

In the past, there were not a lot of studies on how A-kinase anchor protein 5 (AKAP5) involving in the pathogenesis and prognosis of non-mucin producing stomach adenocarcinoma (NMSA). Therefore, we studied the relationship between AKAP5 and the prognosis of NMSA and its possible mechanisms using publicly available data from The Cancer Genome Atlas (TCGA). RNA high-throughput sequencing and clinicopathologic data of NMSA were downloaded from the TCGA. Clinical pathologic features associated with AKAP5 expression were analyzed using the chi-square and Fisher exact tests. The relationship between the overall survival (OS) and AKAP5 expression was analyzed by the Kaplan-Meier method and the Cox regression analysis. GSEA analysis was performed using the TCGA dataset. Our results indicated that the AKAP5 expression was increased in NMSA (all tumor vs. adjacent mucosa). Also, histologic grade, clinical stage, N classification, and survival status were significantly correlated with AKAP5 expression. Kaplan-Meier curves showed that low AKAP5 expression was associated with a poor OS among the NMSA patients (P=5.003e-05), and in the clinical stage III and IV (P=4.646e-05), TNM stage T3 (P=0.016), T4 (P=0.001), N2 (P=0.012), N3 (P=0.003), M0 (P=3.911e-05), and histological grade G3 (P=1.658e-04) subgroups. Cox regression analysis showed that reduced AKAP5 expression in NMSA is associated with age (HR =1.03, P=0.007), stage (HR =1.84 for stage I, II vs. stage III, IV, P=0.002) and M classification (HR =1.8 for M0 vs. M1, P=0.010). Gene sets related to cholesterol homeostasis, glycolysis, estrogen response late, adipogenesis, estrogen response early, notch signaling, and peroxisome were differentially enriched with the low AKAP5 expression phenotype. Low expression of AKAP5 may be a potential molecular marker for predicting poor prognosis of NMSA. Besides, cholesterol homeostasis, glycolysis, estrogen response, adipogenesis, notch signaling, and peroxisome may be the key pathways regulated by AKAP5 in NMSA. It also suggested that AKAP5 might potentially have biological functions in the development of stomach adenocarcinoma.

Polymorphisms/Mutations in A-Kinase Anchoring Proteins (AKAPs): Role in the Cardiovascular System.

A-kinase anchoring proteins (AKAPs) belong to a family of scaffolding proteins that bind to protein kinase A (PKA) by definition and a variety of crucial proteins, including kinases, phosphatases, and phosphodiesterases. By scaffolding these proteins together, AKAPs build a “signalosome” at specific subcellular locations and compartmentalize PKA signaling. Thus, AKAPs are important for signal transduction after upstream activation of receptors ensuring accuracy and precision of intracellular PKA-dependent signaling pathways. Since their discovery in the 1980s, AKAPs have been studied extensively in the heart and have been proven essential in mediating cyclic adenosine monophosphate (cAMP)-PKA signaling. Although expression of AKAPs in the heart is very low, cardiac-specific knock-outs of several AKAPs have a noteworthy cardiac phenotype. Moreover, single nucleotide polymorphisms and genetic mutations in crucial cardiac proteins play a substantial role in the pathophysiology of cardiovascular diseases (CVDs). Despite the significant role of AKAPs in the cardiovascular system, a limited amount of research has focused on the role of genetic polymorphisms and/or mutations in AKAPs in increasing the risk of CVDs. This review attempts to overview the available literature on the polymorphisms/mutations in AKAPs and their effects on human health with a special focus on CVDs.

Suppression of epithelial restitution using an inhibitor against Rho-associated coiled-coil containing protein kinase aggravates colitis through reduced epithelial expression of A-kinase anchor protein 13

In the gastrointestinal tract, the immediate healing response to mucosal damage is critical to sustain mucosal homeostasis. The migration of surrounding epithelial cells to cover the denuded area without proliferation is termed restitution, followed by early reparation of the damage. In this study, we determined the role of A-kinase anchor protein 13 (AKAP13) in mice with dextran sulphate sodium (DSS)-induced colitis upon mucosal injury and restitution, and investigated whether inhibition of Rho-associated coiled-coil containing protein kinase (ROCK), downstream effector of AKAP13, affects these mucosal responses. BALB/c mice were challenged with 4% or 2% DSS in their drinking water for up to 8 or 16days, respectively. During this period, mice received subcutaneous injections of fasudil hydrochloride hydrate (FH, 10mg/kg, twice per day), an inhibitor of phosphorylation of ROCK. In immunohistochemistry, AKAP13 was highly expressed in the mucosal epithelium prior to DSS-induced mucosal injury, and also expressed in ulcer-covering non-proliferative epithelium, which corresponded to restituted epithelial cells. Coadministration of FH increased serum amyloid A levels and histopathological scores for mucosal injury, as compared with the DSS group. The effects were associated with a decrease in gene expression of Akap13 in the mucosal tissue and the inhibition of restitution rata (the length of restituted epithelial cells per ulcer). These results suggested that AKAP13 and ROCK are involved in mucosal response at early injury and restitution during healing in DSS-induced colitis in mice.

Expression of A-kinase anchor protein 13 and Rho-associated coiled-coil containing protein kinase in restituted and regenerated mucosal epithelial cells following mucosal injury and colorectal cancer cells in mouse models

We demonstrate the expression patterns of A-kinase anchor protein 13 (AKAP13), a scaffold protein that acts upstream of Rho signaling, and Rho-associated coiled-coil containing protein kinase (ROCK) 1/2 in mouse colorectal cancer and during the healing stage of mouse colitis. BALB/c mice received an intraperitoneal injection of azoxymethane at 10mg/kg, followed by two 7-day cycles of 3% dextran sulfate sodium (DSS) administered through their drinking water to induce colon cancer, or a 7-day administration of 4% DSS to induce colitis. The colorectal tissue was then analyzed for gene expression, histopathology, and immunohistochemistry. In the colorectal cancer, AKAP13 and ROCK1/2 were highly expressed in adenocarcinoma compared to the control tissue and low-grade dysplasia. In colitis, AKAP13 and ROCK1 were highly expressed in the restituted and regenerated mucosa but were only moderately expressed in the injured mucosal epithelium, compared to the normal epithelium that exhibited weak expression levels. ROCK2 was weakly expressed in these cells, consistent with the expression of AKAP13 and ROCK1. Furthermore, we found several clumps of epithelial cells expressing AKAP13 and ROCK1/2 in the lamina propria during the mucosal healing process, and these cells also expressed interleukin-6, which is a multipotential cytokine for both inflammation and healing. These data suggest that AKAP13 was expressed in relation with ROCK1/2, which probably play an overall role in both mucosal healing and tumorigenesis.

Correlation between the protein expression of A-kinase anchor protein 95, cyclin D3 and AKT and pathological indicators in lung cancer tissues.

The aim of the present study was to investigate the correlation between the protein expression of A-kinase anchor protein 95 (AKAP95), cyclin D3 and AKT with pathological indicators in lung cancer tissues. Immunohistochemistry was used to detect the protein expression levels of the proteins in 51 lung cancer tissue samples and 15 pericarcinoma tissue samples. The percentage of cyclin D3 positive samples in the lung cancer and pericarcinoma tissues was 68.63 and 28.57%, respectively, and the difference was statistically significant (P<0.01). However, cyclin D3 expression was not shown to correlate with differentiation grade, histological type or lymph node metastasis. In addition, the percentage of AKT positive samples in the cancer and pericarcinoma tissues was 76.47 and 38.46%, respectively, and the difference was statistically significant (P<0.05). AKT expression was found to significantly correlate with the grade of cancer tissue differentiation (P<0.05); however, no correlations were observed with histological type or lymph node metastasis (P>0.05). AKAP95 expression was shown to correlate with cyclin D3 and AKT expression in the lung cancer tissue (P<0.05); however, there was no correlation between cyclin D3 and AKT expression. The present study provided evidence suggesting that AKAP95 may have a role in regulation of the cell cycle.

A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle.

β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy.

Cardiac inwardly rectifying IK1 current is regulated by protein kinase A via AKAP15 and AKAP79

Background: Inwardly rectifying potassium current IK1 stabilizes the diastolic resting membrane potential. On the molecular level it is composed by heterotetrameric assembly of Kir2.1, Kir2.2 and Kir2.3 channels. Dysfunction of IK1 current strongly predisposes to focal ventricular ectopy. Protein kinase A (PKA) is a key enzyme in adrenergic signal transduction that is involved in catecholaminergic arrhythmogenesis. Targeted protein phosphorylation through PKA is mediated by A Kinase Anchoring Proteins (AKAPs). To date, however, only little is known about the AKAPs that are of functional relevance for the regulation of cardiac IK1 potassium current. Methods: Cardiac IK1 current was measured in isolated rat ventricular cardiomyocytes using whole-cell patch clamp. Kir2.x channels and AKAPs were expressed in Xenopus oocytes and current recordings were performed using the double-electrode voltage-clamp technique. Association of channels and AKAPs was examined with the use of co-immunoprecipitation and immunofluorescent confocal microscopy in isolated cardiomyocytes and in mammalian cell lines. Results: IK1 current in isolated rat ventricular cardiomyocytes was inhibited by activation of protein kinase A. This regulation was markedly attenuated by disrupting PKA-binding to AKAPs with the peptide Ht31 in the expression system. We observed functional coupling of AKAP15 and AKAP79 to Kir2.1 and Kir2.2 channels, but not to Kir2.3 channels. Kir2.1 channels were only sensitive to PKA regulation if membrane-associated AKAPs were co-expressed. Kir2.2 channels exhibited the strongest sensitivity to PKA regulation that was even further increased after co-expression of AKAPs. By contrast, Kir2.3 channels were insensitive to PKA regulation even after co- expression of AKAPs. When co-expressing the AKAP-IS related inhibitor peptide Ht31 together with AKAPs and Kir2.1 and Kir2.2 channels, PKA- dependent effects were almost completely suppressed. Both AKAP79 and AKAP15 co-immunoprecipitate and co-localize with Kir2.1 and Kir2.2 in cardiomyocytes indicating co-assembly in membrane-bound signalling complexes. Conclusion: In this project, we provide evidence for the functional and biochemical coupling of cardiac Kir2.1 and Kir2.2 channels to the membrane-associated anchoring proteins AKAP15 and AKAP79. Membrane-associated AKAPs may provide novel molecular targets for antiarrhythmic therapy downstream from the cardiac beta1-adrenoreceptor.

Increased density of AKAP5-expressing neurons in the anterior cingulate cortex of subjects with bipolar disorder

Brain anatomical abnormalities as well as cognitive and emotional processing deficits have been reported for the prefrontal cortex in bipolar disorder, which are in part attributable to cellular and laminar abnormalities in postsynaptic protein expression. A kinase anchoring protein (AKAP) 5/79 plays a key role in postsynaptic signalling of excitatory synapses. We aimed to reveal if the cellular expression of AKAP5/79 protein is altered in the anterior cingulate cortex and the dorsolateral prefrontal cortex in bipolar disorder. Ten subjects with bipolar disorder and ten control cases were investigated by use of immunohistochemical and morphometric techniques. Compared with controls in subjects with bipolar disorder, the numerical density of AKAP5-expressing neurons was significantly increased in the left (p = 0.002) and right (p = 0.008) anterior cingulate cortex. Layer-specific counting revealed that left side layers II (p = 0.000), III (p = 0.001) and V (p = 0.005) as well as right side layers III (p = 0.007), IV (p = 0.007) and V (p = 0.004) had significantly increased AKAP5-positive cell densities in bipolar disorder. In contrast, no statistically significant differences were found for the dorsolateral prefrontal cortex. However, we observed a more intense intraneuronal immunostaining in both prefrontal areas in bipolar disorder patients. Elevated cell numbers and increased intracellular expression of AKAP, together with the altered expression patterns of most intracellular interaction partners of this protein in bipolar disorder as known from the literature, might point to disease-related abnormalities of the AKAP-associated signalosome in prefrontal cortex neurons.

Expression of a kinase anchoring protein 79 in the human fetal amygdala.

The expression of AKAP (a kinase anchoring protein) 79 enriched in postsynaptic densities has been investigated in the human amygdaloid nuclei of the 8th gestational month. Nuclear specific diffuse and cellular immunostaining is observed. An outstanding feature of cellular immunostaining is the labelling of somata and dendritic trees in a manner that allows neuronal classification. Bipolar, multipolar, and pyramidal AKAP79-positive neurons are found throughout the amygdala; the highest packing density of immunostained neurons is seen within the central and lateral nucleus. Dense diffuse immunolabelling is observed in the lateral and accessory basal nucleus. The results indicate that AKAP79 is expressed in various neuronal types (projection as well as local circuit neurons). Diffuse staining does not always match with cellular labelling within a nucleus, thus, AKAP79 may be particularly enriched in dendrites in some nuclei. The widespread distribution of AKAP79 indicates its possible role in various amygdaloid circuitries; thus, AKAP79 does not seem to be restricted to definite functional systems in the 8th month.

Expression of A Kinase Anchor Protein 121 Is Regulated by Hormones in Thyroid and Testicular Germ Cells

Distinct A Kinase Anchor Proteins (AKAPs) immobilize and concentrate protein kinase A II (PKAII) isoforms at specific intracellular locations. AKAP121 binds and targets PKAIIalpha to the cytoplasmic surface of mitochondria. Mechanisms that control expression of this mitochondrial AKAP are unknown. We have cloned cDNA for rat AKAP121 and show that AKAP121 protein expression is regulated by thyroid stimulating hormone (TSH) and cAMP. Differentiated thyroid cells (TL5) accumulate AKAP121 upon incubation with TSH or a cAMP analog. Levels of total and newly synthesized AKAP121 mRNA also increased after treatment. AKAP121 mRNA accumulated in the presence of cycloheximide, suggesting that transcription of the anchor protein gene is directly controlled by cAMP and PKA. AKAP121 is induced with similar kinetics when an unrelated, spermatocyte-derived cell line (GC-2) is incubated with 8-chlorophenylthio-cAMP. Thus, AKAP121 concentration may be controlled by hormones that activate adenylate cyclase. This mode of regulation could provide a general mechanism for (a) enhancing the sensitivity of distal organelles to cAMP and (b) shifting the focus of cAMP-mediated signaling from cytoplasm to organelles.

Regulation of expression of A-kinase anchoring proteins in rat granulosa cells.

FSH action on granulosa cells involves the generation of cAMP and subsequent activation of the cAMP-dependent protein kinase (PKA). The PKA holoenzyme is targeted to specific subcellular sites through the interaction of the regulatory subunits with A-kinase anchoring proteins (AKAPs). We previously reported that FSH regulates expression of AKAPs. In this report we examine the relationship between AKAP expression and cell shape. Granulosa cells cultured in the absence of FSH tend to spread and flatten. Cell spreading is accompanied by an increased expression of a 140-kDa AKAP. This spreading/flattening phenotype is independent of the specific extracellular matrix proteins (fibronectin, polylysine, and gelatin) on which cells are plated. Addition of FSH prevents both cell spreading and induction of AKAP 140. Culturing cells on poly (2-hydroxyethyl methacrylate), a surface-coating agent that inhibits cell spreading and adhesion, also inhibits expression of AKAP 140. Addition of phorbol myristate acetate, an agent known to antagonize FSH actions, blocks FSH regulation of both cell shape and AKAP 140 expression. Addition of dexamethasone plus FSH causes a synergistic increase in progesterone levels but has no effect on cell shape or induction of AKAP 140. Dexamethasone produces a dose-dependent increase in AKAP 80 expression, which is blocked by FSH, suggesting cross talk between the glucocorticoid and FSH receptor signaling pathways. These data suggest that expression of AKAP 140 is linked to regulation of cell shape, and that changes in the expression of AKAPs are regulated by several different signaling pathways.

Expression of a kinase anchor protein 75 depletes type II cAMP-dependent protein kinases from the cytoplasm and sequesters the kinases in a particulate pool.

A kinase anchor proteins (AKAPs) have a C-terminal binding site for the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (PKAII beta) and an N-terminal domain that mediates the targeting and attachment of the anchor protein to intracellular structures. In vitro biochemical studies and recent in situ immunocytochemical analysis (Glantz, S. B., Amat, J. A., and Rubin, C. S. (1992) Mol. Biol. Cell 3, 1215-1228) suggest that AKAPs anchor PKAII beta at specific sites in the dendritic cytoskeleton of neurons. This arrangement would place PKAII beta in proximity with its substrates and create "target sites" for cAMP actions. The foregoing model predicts that (a) RII subunits are freely accessible to AKAPs, (b) PKAII holoenzymes, as well as RII subunits, are anchored, and (c) changes in the level of AKAP can alter the intracellular distribution of type II PKAs. We have addressed these previously untested propositions by overexpressing bovine AKAP75 in a human cell line (HEK293). Non-transfected cells express a low level of endogenous AKAP79, and approximately 90% of RII alpha and RII beta subunits are isolated in the cell cytosol. In contrast, stably transfected cells, which express a 10-fold excess of AKAP75, sequester > 90% of their RII subunits in a particulate pool. Catalytic subunits are also transferred to this pool. AKAP75 accumulates in a cell compartment with biochemical properties characteristic of cytoskeleton. Thus, AKAPs have access to and avidly bind cytoplasmic type II PKAs. Moreover, an increase in AKAP content can alter the particulate/cytoplasmic distribution of PKAII beta and PKAII alpha.