RESUMEN
Triple-negative breast cancers are the most aggressive subtypes with poor prognosis due to lack of targeted cancer therapy. Recently, we reported an association of A-kinase anchor protein 4 expression with various clinico-pathological parameters of breast cancer patients. In this context, we examined the effect of knockdown of A-kinase anchor protein 4 on cell cycle, apoptosis, cellular proliferation, colony formation, migration, and invasion in triple-negative breast cancer cells. We also examined the synergistic cytotoxic effect of paclitaxel on A-kinase anchor protein 4 downregulated triple-negative breast cancer cells. Knockdown of A-kinase anchor protein 4 resulted in significant reduction in cellular growth and migratory abilities. Interestingly, we also observed enhanced cell death in A-kinase anchor protein 4 downregulated cells treated with paclitaxel. Knockdown of A-kinase anchor protein 4 in cell cycle resulted in G0/G1 phase arrest. Knockdown of A-kinase anchor protein 4 also led to increased reactive oxygen species generation as a result of upregulation of NOXA and CHOP. In addition, levels of cyclins, cyclin-dependent kinases, anti-apoptotic molecules, and mesenchymal markers were reduced in A-kinase anchor protein 4 downregulated cells. Moreover, downregulation of A-kinase anchor protein 4 also caused tumor growth reduction in in vivo studies. These data together suggest that A-kinase anchor protein 4 downregulation inhibits various malignant properties and enhances the cytotoxic effect of paclitaxel, and this combinatorial approach could be useful for triple-negative breast cancer treatment.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Neoplasias de la Mama Triple Negativas/genética , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Biomarcadores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Inmunofenotipificación , Ratones , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Paclitaxel/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gravin, an A-kinase anchoring protein, is known to play a role in regulating key processes that lead to inflammation and atherosclerosis development, namely, cell migration, proliferation, and apoptosis. We investigated the role of gravin in the development of high-fat diet (HFD)-induced atherosclerosis and hyperlipidemia. Five-week-old male wild-type (WT) and gravin-t/t mice were fed a normal diet or an HFD for 16 wk. Gravin-t/t mice showed significantly lower liver-to-body-weight ratio, cholesterol, triglyceride, and very low-density lipoprotein levels in serum as compared with WT mice on HFD. Furthermore, there was less aortic plaque formation coupled with decreased lipid accumulation and liver damage, as the gravin-t/t mice had lower levels of serum alanine aminotransferase and aspartate aminotransferase. Additionally, gravin-t/t HFD-fed mice had decreased expression of liver 3-hydroxy-3-methyl-glutaryl-CoA reductase, an essential enzyme for cholesterol synthesis and lower fatty acid synthase expression. Gravin-t/t HFD-fed mice also exhibited inhibition of sterol regulatory element binding protein-2 (SREBP-2) expression, a liver transcription factor associated with the regulation of lipid transportation. In response to platelet-derived growth factor receptor treatment, gravin-t/t vascular smooth muscle cells exhibited lower intracellular calcium transients and decreased protein kinase A- and protein kinase C-dependent substrate phosphorylation, notably involving the Erk1/2 signaling pathway. Collectively, these results suggest the involvement of gravin-dependent regulation of lipid metabolism via the reduction of SREBP-2 expression. The absence of gravin-mediated signaling lowers blood pressure, reduces plaque formation in the aorta, and decreases lipid accumulation and damage in the liver of HFD mice. Through these processes, the absence of gravin-mediated signaling complex delays the HFD-induced hyperlipidemia and atherosclerosis.NEW & NOTEWORTHY The gravin scaffolding protein plays a key role in the multiple enzymatic pathways of lipid metabolism. We have shown for the first time the novel role of gravin in regulating the pathways related to the initiation and progression of atherosclerosis. Specifically, an absence of gravin-mediated signaling decreases the lipid levels (cholesterol, triglyceride, and VLDL) that are associated with sterol regulatory element binding protein-2 downregulation.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Proteínas de Ciclo Celular/deficiencia , Dieta Alta en Grasa , Hiperlipidemias/prevención & control , Lípidos/sangre , Placa Aterosclerótica , Proteínas de Anclaje a la Quinasa A/genética , Animales , Aorta/patología , Enfermedades de la Aorta/sangre , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Aterosclerosis/sangre , Aterosclerosis/etiología , Aterosclerosis/genética , Proteínas de Ciclo Celular/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hiperlipidemias/sangre , Hiperlipidemias/etiología , Hiperlipidemias/genética , Hígado/enzimología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Transducción de Señal , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
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.
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Proteínas de Anclaje a la Quinasa A/biosíntesis , Neocórtex/metabolismo , Corteza Somatosensorial/metabolismo , Sinapsis/metabolismo , Tálamo/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Animales , Expresión Génica , Ratones , Ratones Noqueados , Ratones Transgénicos , Neocórtex/citología , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Corteza Somatosensorial/citología , Sinapsis/genética , Tálamo/citologíaRESUMEN
Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.
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Infertilidad Masculina/genética , Mutación con Pérdida de Función , Proteínas de Microtúbulos/genética , Cola del Espermatozoide/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Adulto , Animales , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Consanguinidad , Expresión Génica , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Noqueados , Linaje , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Motilidad Espermática , Cola del Espermatozoide/patología , Cola del Espermatozoide/ultraestructura , Testículo/química , Testículo/metabolismo , Secuenciación Completa del GenomaRESUMEN
OBJECTIVE: Hormonal contributions to the sex-dependent development of both obsessive-compulsive disorder (OCD) and obesity have been described, but the underlying mechanisms are incompletely understood. A-kinase anchoring protein 13 (AKAP13) significantly augments ligand-dependent activation of estrogen receptors alpha and beta. The hypothalamus and pituitary gland are implicated in the development and exacerbation of OCD and obesity and have strong AKAP13 expression. The AKAP13 localization pattern observed in these key brain regions together with its effects on sex steroid action suggest a potential role for AKAP13 in compulsive-like behaviors. Here we tested the role of AKAP13 in compulsive-like behavior and body weight using an Akap13 haploinsufficient murine model. MATERIALS AND METHODS: Targeted deletion of the Akap13 gene generated haploinsufficient (Akap13+/-) mice in a C57BL6/J genetic background. Established behavioral assays were conducted, video recorded, and scored blindly to assess compulsive-like behavior based on genotype and gender. Tests included: marble-burying, grooming, open- field and elevated plus-maze. Brain and body weights were also obtained. Mean levels of test outcomes were compared using multi-way ANOVA to test for genotype, sex, genotype*sex, and genotype*sex*age interaction effects with Bonferroni adjustment for multiple comparisons, to further explain any significant interactions. RESULTS: The marble-burying and grooming assays revealed significant sex-dependent increases in perseverative, compulsive-like behaviors in female Akap13 haploinsufficient mice compared to female wild type (WT) mice by demonstrating increased marble-burying activity (pâ¯=â¯.0025) and a trend towards increased grooming behavior (pâ¯=â¯.06). Male Akap13 haploinsufficient mice exhibited no behavioral changes (pâ¯>â¯0.05). Elevated plus-maze and open-field test results showed no overt anxiety-like behavior in Akap13 haploinsufficient mice irrespective of sex (pâ¯>â¯0.05, both). No differences in brain weight were found in Akap13 haploinsufficient mice compared to WT mice (pâ¯>â¯0.05). However, female Akap13 haploinsufficient mice weighed more than female WT mice in the 4 to <7 months age range (pâ¯=â¯.0051). Male Akap13 haploinsufficient mice showed no differences in weight compared to male WT mice (pâ¯=â¯>0.05) at any age range examined. CONCLUSION: Akap13 haploinsufficiency led to sex-dependent, compulsive-like behavioral changes in a murine model. Interestingly, Akap13 haploinsufficiency also led to a sex-dependent increase in body weight. These results revealed a requirement for AKAP13 in murine behavior, particularly in female mice, and is the first report of AKAP13 involvement in murine behavior. Future studies may examine the involvement of AKAP13 in the pathophysiology of OCD in female humans and may contribute to a better understanding of the role of AKAP13 and sex hormones in the development and exacerbation of OCD.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/deficiencia , Peso Corporal/fisiología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Trastorno Obsesivo Compulsivo/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Animales , Ansiedad/metabolismo , Conducta Animal/fisiología , Conducta Compulsiva/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor/genética , Obesidad/metabolismo , Factores SexualesRESUMEN
Hypertension is a multifactorial chronic inflammatory disease that leads to cardiac remodeling. A-kinase anchor protein 12 (AKAP12) is a scaffolding protein that has multiple functions in various biological events, including the regulation of vessel integrity and differentiation of neural barriers in blood. However, the role of AKAP12 in angiotensin II (Ang II)-induced cardiac injury remains unclear. In the present study, Ang II infusion reduced AKAP12 expressions in the hearts of wild-type (WT) mice, and AKAP12 knockout (KO) enhanced the infiltration of inflammatory cells. In addition, AKAP12 deletion accelerated Ang II-induced cardiac histologic alterations and dysfunction. Further, AKAP12-/- aggravated heart failure by promoting the inflammation, oxidative stress, cellular apoptosis, and autophagy induced by Ang II. Furthermore, AKAP12 KO elevated Ang II-induced cardiac fibrosis, as indicated by the following: (1) Masson trichrome staining showed that Ang II infusion markedly increased fibrotic areas of the WT mouse heart, which was greatly accelerated in AKAP12-/- mice; (2) immunohistochemistry analysis showed increased expression of transforming growth factor ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) in the AKAP12-/- mouse heart; (3) reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed increased expression of fibrosis-related molecules in the AKAP12-deficient mouse heart; and (4) Western blot analysis indicated significantly higher upregulation of p-SMAD2/3 in the AKAP12-/- mouse heart. In vitro, AKAP12 knockdown in HL-1â¯cells was responsible for TGF-ß1-induced inflammation, the generation of reactive oxygen species (ROS), apoptosis, autophagy, and fibrosis. Furthermore, overexpression of AKAP12 reduced fibrosis triggered by TGF-ß1 in cells. Overall, our study suggests that fibrosis induced by Ang II may be alleviated by AKAP12 expression through inactivation of the TGF-ß1 pathway.
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Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Lesiones Cardíacas/metabolismo , Lesiones Cardíacas/patología , Miocardio/patología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/metabolismo , Angiotensina II , Animales , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Progresión de la Enfermedad , Fibrosis , Técnicas de Silenciamiento del Gen , Lesiones Cardíacas/inducido químicamente , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
AIMS: Impaired Ca2 + cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca2+ cycling and excitation-contraction in cardiomyocytes. However, the role of the AKAP150 signalling complexes in the pathogenesis of heart failure has not been investigated. METHODS AND RESULTS: Here we examined how AKAP150 signalling complexes impact Ca2+ cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodelling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not affect calcineurin-nuclear factor of activated T cells signalling in cardiomyocytes or pressure overload- or agonist-induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca2+ regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca2+ cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. CONCLUSIONS: These findings define a critical role for AKAP150 in regulating Ca2+ cycling and myocardial ionotropy following pathological stress, suggesting the AKAP150 signalling pathway may serve as a novel therapeutic target for heart failure.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Señalización del Calcio , Cardiomiopatía Dilatada/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocitos Cardíacos/metabolismo , Función Ventricular , Remodelación Ventricular , Proteínas de Anclaje a la Quinasa A/genética , Animales , Calcineurina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/patología , Cardiomiopatía Dilatada/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis , Predisposición Genética a la Enfermedad , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Isoproterenol , Ratones Noqueados , Isquemia Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/complicaciones , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Fenotipo , Interferencia de ARN , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Protein Kinase A (PKA) mediates synaptic plasticity and is widely implicated in learning and memory. The hippocampal dentate gyrus (DG) is thought to be responsible for processing and encoding distinct contextual associations in response to highly similar inputs. The mossy fiber (MF) axons of the dentate granule cells convey strong excitatory drive to CA3 pyramidal neurons and express presynaptic, PKA-dependent forms of plasticity. Here, we demonstrate an essential role for the PKA anchoring protein, AKAP7, in mouse MF axons and terminals. Genetic ablation of AKAP7 specifically from dentate granule cells results in disruption of MF-CA3 LTP directly initiated by cAMP, and the AKAP7 mutant mice are selectively deficient in pattern separation behaviors. Our results suggest that the AKAP7/PKA complex in the MF projections plays an essential role in synaptic plasticity and contextual memory formation.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Región CA3 Hipocampal/fisiología , Núcleos Cerebelosos/fisiología , Técnicas de Inactivación de Genes , Fibras Musgosas del Hipocampo/fisiología , Conducta Espacial , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , RatonesRESUMEN
A muscle-specific nonkinase anchoring protein (αkap), encoded within the calcium/calmodulin kinase II (camk2) α gene, was recently found to control the stability of acetylcholine receptor (AChR) clusters on the surface of cultured myotubes. However, it remains unknown whether this protein has any effect on receptor stability and the maintenance of the structural integrity of neuromuscular synapses in vivo. By knocking down the endogenous expression of αkap in mouse sternomastoid muscles with shRNA, we found that the postsynaptic receptor density was dramatically reduced, the turnover rate of receptors at synaptic sites was significantly increased, and the insertion rates of both newly synthesized and recycled receptors into the postsynaptic membrane were depressed. Moreover, we found that αkap shRNA knockdown impaired synaptic structure as postsynaptic AChR clusters and their associated postsynaptic scaffold proteins within the neuromuscular junction were completely eliminated. These results provide new mechanistic insight into the role of αkap in regulating the stability of the postsynaptic apparatus of neuromuscular synapses.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Unión Neuromuscular/metabolismo , Densidad Postsináptica/metabolismo , Receptores Colinérgicos/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/efectos de los fármacos , Ratones , Músculos del Cuello/metabolismo , Densidad Postsináptica/efectos de los fármacos , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/farmacologíaRESUMEN
Mechanical stimulation is crucial to bone growth and triggers osteogenic differentiation through a process involving Rho and protein kinase A. We previously cloned a gene (AKAP13, aka BRX) encoding a protein kinase A-anchoring protein in the N-terminus, a guanine nucleotide-exchange factor for RhoA in the mid-section, coupled to a carboxyl region that binds to estrogen and glucocorticoid nuclear receptors. Because of the critical role of Rho, estrogen, and glucocorticoids in bone remodeling, we examined the multifunctional role of Akap13. Akap13 was expressed in bone, and mice haploinsufficient for Akap13 (Akap13(+/-)) displayed reduced bone mineral density, reduced bone volume/total volume, and trabecular number, and increased trabecular spacing; resembling the changes observed in osteoporotic bone. Consistent with the osteoporotic phenotype, Colony forming unit-fibroblast numbers were diminished in Akap13(+/-) mice, as were osteoblast numbers and extracellular matrix production when compared to control littermates. Transcripts of Runx2, an essential transcription factor for the osteogenic lineage, and alkaline phosphatase (Alp), an indicator of osteogenic commitment, were both reduced in femora of Akap13(+/-) mice. Knockdown of Akap13 reduced levels of Runx2 and Alp transcripts in immortalized bone marrow stem cells. These findings suggest that Akap13 haploinsufficient mice have a deficiency in early osteogenesis with a corresponding reduction in osteoblast number, but no impairment of mature osteoblast activity.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Densidad Ósea , Factores de Intercambio de Guanina Nucleótido/deficiencia , Osteoporosis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Haploinsuficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
AIMS: Cardiac ß-adrenergic receptors (ß-AR) are key regulators of cardiac haemodynamics and size. The scaffolding protein A-kinase anchoring protein 79/150 (AKAP5) is a key regulator of myocardial signalling by ß-ARs. We examined the function of AKAP5 in regulating cardiac haemodynamics and size, and the role of ß-ARs and Ca(2+)-regulated intracellular signalling pathways in this phenomenon. METHODS AND RESULTS: We used echocardiographic, histological, genetic, and biochemical methods to examine the effect of ablation of AKAP5 on cardiac haemodynamics, size, and signalling in mice. AKAP5(-/-) mice exhibited enhanced signs of cardiac dilatation and dysfunction that progressed with age. Infusions of isoprenaline worsened cardiac haemodynamics in wild-type (WT) mice only, but increased the ratio of heart-to-body weight equally in WT and in AKAP5(-/-) mice. Mechanistically, loss of AKAP5 was associated with enhanced activity of cardiac calmodulin kinase II (CaMKII) and calcineurin (CaN) as indexed by nuclear factor of activated T-cell-luciferase activity. Loss of AKAP5 interfered with the recycling of cardiac ß1-ARs, which was mediated in part by CaN binding to AKAP5. Carvedilol reversed cardiac hypertrophy and haemodynamic deficiencies in AKAP5(-/-) mice by normalizing the activities of cardiac CaN and CaMKII. CONCLUSIONS: These findings identify a novel cardioprotective role for AKAP5 that is mediated by regulating the activities of cardiac CaN and CaMKII and highlight a significant role for cardiac ß-ARs in this phenomenon.
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Proteínas de Anclaje a la Quinasa A/deficiencia , Antagonistas Adrenérgicos/farmacología , Calcineurina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Carbazoles/farmacología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Miocitos Cardíacos/efectos de los fármacos , Propanolaminas/farmacología , Disfunción Ventricular Izquierda/tratamiento farmacológico , Función Ventricular Izquierda/efectos de los fármacos , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Carvedilol , Células Cultivadas , Hemodinámica/efectos de los fármacos , Hipertrofia Ventricular Izquierda/enzimología , Hipertrofia Ventricular Izquierda/genética , Hipertrofia Ventricular Izquierda/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/metabolismo , Ratas Sprague-Dawley , Receptores Adrenérgicos beta 1/efectos de los fármacos , Receptores Adrenérgicos beta 1/metabolismo , Factores de Tiempo , Disfunción Ventricular Izquierda/enzimología , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Neurobeachin (Nbea) is implicated in vesicle trafficking in the regulatory secretory pathway, but details on its molecular function are currently unknown. We have used Drosophila melanogaster mutants for rugose (rg), the Drosophila homolog of Nbea, to further elucidate the function of this multidomain protein. Rg is expressed in a granular pattern reminiscent of the Golgi network in neuronal cell bodies and colocalizes with transgenic Nbea, suggesting a function in secretory regulation. In contrast to Nbea(-/-) mice, rg null mutants are viable and fertile and exhibit aberrant associative odor learning, changes in gross brain morphology, and synaptic architecture as determined at the larval neuromuscular junction. At the same time, basal synaptic transmission is essentially unaffected, suggesting that structural and functional aspects are separable. Rg phenotypes can be rescued by a Drosophila rg+ transgene, whereas a mouse Nbea transgene rescues aversive odor learning and synaptic architecture; it fails to rescue brain morphology and appetitive odor learning. This dissociation between the functional redundancy of either the mouse or the fly transgene suggests that their complex composition of numerous functional and highly conserved domains support independent functions. We propose that the detailed compendium of phenotypes exhibited by the Drosophila rg null mutant provided here will serve as a test bed for dissecting the different functional domains of BEACH (for beige and human Chediak-Higashi syndrome) proteins, such as Rugose, mouse Nbea, or Nbea orthologs in other species, such as human.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/fisiología , Aprendizaje por Asociación/fisiología , Encéfalo/citología , Proteínas de Drosophila/fisiología , Sinapsis/fisiología , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Análisis de Varianza , Animales , Animales Modificados Genéticamente , Encéfalo/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Drosophila , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Peroxidasa de Rábano Silvestre/metabolismo , Masculino , Potenciales de la Membrana/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/citología , Unión Neuromuscular/genética , Neuronas/citología , Odorantes , Neuronas Receptoras Olfatorias/citología , Técnicas de Placa-Clamp , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , ARN Mensajero/metabolismo , Estadísticas no Paramétricas , Sinapsis/genéticaRESUMEN
Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic ß-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-ß-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in ß-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/fisiología , Glucosa/metabolismo , Homeostasis/fisiología , Resistencia a la Insulina/genética , Proteínas de la Membrana/fisiología , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Anclaje a la Quinasa A/química , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Secuencias de Aminoácidos , Animales , Calcineurina/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , AMP Cíclico/fisiología , Glucosa/farmacología , Homeostasis/efectos de los fármacos , Insulina/metabolismo , Insulina/farmacología , Secreción de Insulina , Insulinoma/patología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Hígado/enzimología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Músculo Esquelético/enzimología , Neoplasias Pancreáticas/patología , Mapeo de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/fisiología , Eliminación de Secuencia , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIalpha or RIIbeta as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and beta-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/genética , Dendritas/patología , Fenómenos Electrofisiológicos/genética , Discapacidades para el Aprendizaje/genética , Trastornos de la Memoria/genética , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/fisiología , Animales , Sitios de Unión , Cuerpo Estriado , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dendritas/metabolismo , Hipocampo , Discapacidades para el Aprendizaje/etiología , Trastornos de la Memoria/etiología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Plasticidad Neuronal , Unión Proteica , Transducción de SeñalRESUMEN
A fundamental biologic principle is that diverse biologic signals are channeled through shared signaling cascades to regulate development. Large scaffold proteins that bind multiple proteins are capable of coordinating shared signaling pathways to provide specificity to activation of key developmental genes. Although much is known about transcription factors and target genes that regulate cardiomyocyte differentiation, less is known about scaffold proteins that couple signals at the cell surface to differentiation factors in developing heart cells. Here we show that AKAP13 (also known as Brx-1, AKAP-Lbc, and proto-Lbc), a unique protein kinase A-anchoring protein (AKAP) guanine nucleotide exchange region belonging to the Dbl family of oncogenes, is essential for cardiac development. Cardiomyocytes of Akap13-null mice had deficient sarcomere formation, and developing hearts were thin-walled and mice died at embryonic day 10.5-11.0. Disruption of Akap13 was accompanied by reduced expression of Mef2C. Consistent with a role of AKAP13 upstream of MEF2C, Akap13 siRNA led to a reduction in Mef2C mRNA, and overexpression of AKAP13 augmented MEF2C-dependent reporter activity. The results suggest that AKAP13 coordinates Galpha(12) and Rho signaling to an essential transcription program in developing cardiomyocytes.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Corazón Fetal/embriología , Corazón Fetal/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Anclaje a la Quinasa A/antagonistas & inhibidores , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Corazón Fetal/anomalías , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Hibridación in Situ , Factores de Transcripción MEF2 , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Antígenos de Histocompatibilidad Menor , Modelos Cardiovasculares , Datos de Secuencia Molecular , Miocitos Cardíacos/metabolismo , Factores Reguladores Miogénicos/genética , Factores Reguladores Miogénicos/metabolismo , Embarazo , ARN Interferente Pequeño/genética , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Extracellular hyperosmolarity, or osmotic stress, generally caused by differences in salt and macromolecule concentrations across the plasma membrane, occurs in lymphoid organs and at inflammatory sites. The response of immune cells to osmotic stress is regulated by nuclear factor of activated T cells 5 (NFAT5), a transcription factor that induces the expression of hyperosmolarity-responsive genes and stimulates cytokine production. We report that the guanine nucleotide exchange factor (GEF) Brx [also known as protein kinase A-anchoring protein 13 (AKAP13)] is essential for the expression of nfat5 in response to osmotic stress, thus transmitting the extracellular hyperosmolarity signal and enabling differentiation of splenic B cells and production of immunoglobulin. This process required the activity of p38 mitogen-activated protein kinase (MAPK) and NFAT5 and involved a physical interaction between Brx and c-Jun N-terminal kinase (JNK)-interacting protein 4 (JIP4), a scaffold molecule specific to activation of the p38 MAPK cascade. Our results indicate that Brx integrates the responses of immune cells to osmotic stress and inflammation by elevating intracellular osmolarity and stimulating the production of cytokines.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/fisiología , Linfocitos/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Animales , Factor Activador de Células B/sangre , Linfocitos B/inmunología , Deshidratación , Regulación de la Expresión Génica , Humanos , Inmunoglobulinas/biosíntesis , Linfocitos/enzimología , Linfocitos/inmunología , Ratones , Ratones Noqueados , Antígenos de Histocompatibilidad Menor , Presión Osmótica , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Bazo/anatomía & histología , Bazo/enzimología , Bazo/fisiología , Factores de Transcripción/genéticaRESUMEN
A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate context-dependent neuronal signaling events in vivo.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/deficiencia , Neuronas/fisiología , Animales , Cerebelo/citología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hipocampo/citología , Ratones , Ratones Noqueados , Trastornos de la Destreza Motora/etiología , Agonistas Muscarínicos/farmacología , Proteínas del Tejido Nervioso , Receptores AMPA/metabolismo , Convulsiones , Transducción de Señal/fisiologíaRESUMEN
Certain phosphorylation events are tightly controlled by scaffolding proteins such as A-kinase anchoring protein (AKAP). On nociceptive terminals, phosphorylation of transient receptor potential channel type 1 (TRPV1) results in the sensitization to many different stimuli, contributing to the development of hyperalgesia. In this study, we investigated the functional involvement of AKAP150 in mediating sensitization of TRPV1, and found that AKAP150 is co-expressed in trigeminal ganglia (TG) neurons from rat and associates with TRPV1. Furthermore, siRNA-mediated knock-down of AKAP150 expression led to a significant reduction in PKA phosphorylation of TRPV1 in cultured TG neurons. In CHO cells, the PKA RII binding site on AKAP was necessary for PKA enhancement of TRPV1-mediated Ca2+-accumulation. In addition, AKAP150 knock-down in cultured TG neurons attenuated PKA sensitization of TRPV1 activity and in vivo administration of an AKAP antagonist significantly reduced prostaglandin E2 sensitization to thermal stimuli. These data suggest that AKAP150 functionally regulates PKA-mediated phosphorylation/sensitization of the TRPV1 receptor.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Hiperalgesia/metabolismo , Canales Catiónicos TRPV/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/fisiología , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Cobayas , Hiperalgesia/enzimología , Neuronas/enzimología , Neuronas/metabolismo , Fosforilación , ARN Interferente Pequeño/fisiología , Ratas , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Ganglio del Trigémino/citología , Ganglio del Trigémino/enzimología , Ganglio del Trigémino/metabolismoRESUMEN
Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas/metabolismo , Espermatozoides/metabolismo , Proteínas de Anclaje a la Quinasa A/deficiencia , Proteínas de Anclaje a la Quinasa A/genética , Animales , Bicarbonatos/farmacología , Masculino , Ratones , Ratones Noqueados , Peso Molecular , Fosforilación , Proteínas/química , Transducción de Señal/efectos de los fármacos , Capacitación Espermática , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/efectos de los fármacos , Especificidad por SustratoRESUMEN
Hypertension is a perplexing multiorgan disease involving renal primary pathology and enhanced angiotensin II vascular reactivity. Here, we report that a novel form of a local Ca2+ signaling in arterial smooth muscle is linked to the development of angiotensin II-induced hypertension. Long openings and reopenings of L-type Ca2+ channels in arterial myocytes produce stuttering persistent Ca2+ sparklets that increase Ca2+ influx and vascular tone. These stuttering persistent Ca2+ sparklets arise from the molecular interactions between the L-type Ca2+ channel and protein kinase Calpha at only a few subsarcolemmal regions in resistance arteries. We have identified AKAP150 as the key protein, which targets protein kinase Calpha to the L-type Ca2+ channels and thereby enables its regulatory function. Accordingly, AKAP150 knockout mice (AKAP150-/-) were found to lack persistent Ca2+ sparklets and have lower arterial wall intracellular calcium ([Ca2+]i) and decreased myogenic tone. Furthermore, AKAP150-/- mice were hypotensive and did not develop angiotensin II-induced hypertension. We conclude that local control of L-type Ca2+ channel function is regulated by AKAP150-targeted protein kinase C signaling, which controls stuttering persistent Ca2+ influx, vascular tone, and blood pressure under physiological conditions and underlies angiotensin II-dependent hypertension.