scholarly journals Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A

2014 ◽  
Vol 307 (3) ◽  
pp. C266-C277 ◽  
Author(s):  
Shaima Salman ◽  
Alison C. Holloway ◽  
Colin A. Nurse
Keyword(s):  
Protein Kinase ◽  
Carbonic Anhydrase ◽  
Protein Kinase A ◽  
Chromaffin Cells ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Neonatal Rat ◽  
Pcr Analysis ◽  
Kinase A

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K+ (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ {[d-Arg2,Ly4]dermorphin-(1–4)-amide}- and δ ([d-Pen2,5,P-Cl-Phe4]enkephalin)-opioid agonists (2 μM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

Molecular mechanisms for protein kinase A-mediated modulation of immune function

2002 ◽  
Vol 14 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Knut Martin Torgersen ◽  
Torkel Vang ◽  
Hilde Abrahamsen ◽  
Sheraz Yaqub ◽  
Kjetil Taskén
Keyword(s):  
Protein Kinase ◽  
Immune Function ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Kinase A

scholarly journals Characterization of Proteins Regulated by Androgen and Protein Kinase a Signaling in VCaP Prostate Cancer Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1404
Author(s):  
Hye-Jin You ◽  
Byong-Chul You ◽  
Jong-Kwang Kim ◽  
Jae-Min Park ◽  
Bo-Seul Song ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Cancer Patients ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Castration Resistant Prostate Cancer ◽  
Normal Prostate ◽  
Prostate Cancer Development ◽  
Kinase A

Androgen signaling via the androgen receptor (AR) is involved in normal prostate development and prostate cancer progression. In addition to androgen binding, a variety of protein kinases, including cyclic AMP-dependent protein kinase A (PKA), can activate the AR. Although hormone deprivation, especially that of androgen, continues to be an important strategy for treating prostate cancer patients, the disease ultimately progresses to castration-resistant prostate cancer (CRPC), despite a continuous hormone-deprived environment. To date, it remains unclear which pathways in this progression are active and targetable. Here, we performed a proteomic analysis of VCaP cells stimulated with androgen or forskolin to identify proteins specific for androgen-induced and androgen-bypassing signaling, respectively. Patterns of differentially expressed proteins were quantified, and eight proteins showing significant changes in expression were identified. Functional information, including a Gene Ontology analysis, revealed that most of these proteins are involved in metabolic processes and are associated with cancer. The mRNA and protein expression of selected proteins was validated, and functional correlations of identified proteins with signaling in VCaP cells were assessed by measuring metabolites related to each enzyme. These analyses offered new clues regarding effector molecules involved in prostate cancer development, insights that are supported by the demonstration of increased expression levels of the eight identified proteins in prostate cancer patients and assessments of the progression-free interval. Taken together, our findings show that aberrant levels of eight proteins reflect molecular changes that are significantly regulated by androgen and/or PKA signaling pathways, suggesting possible molecular mechanisms of CRPC.

scholarly journals Role of Calcium and EPAC in Norepinephrine-Induced Ghrelin Secretion

Endocrinology ◽  
2014 ◽  
Vol 155 (1) ◽  
pp. 98-107 ◽  
Author(s):  
Bharath K. Mani ◽  
Jen-Chieh Chuang ◽  
Lilja Kjalarsdottir ◽  
Ichiro Sakata ◽  
Angela K. Walker ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Intracellular Signaling ◽  
Endocrine Cells ◽  
Molecular Mechanisms ◽  
Phosphodiesterase Inhibitor ◽  
Intracellular Signaling Pathways ◽  
Ghrelin Secretion ◽  
Kinase A

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to β1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca2+ and cAMP. Several voltage-gated Ca2+ channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca2+ levels both in the presence and absence of extracellular Ca2+. Ca2+-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca2+ influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca2+. Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.

scholarly journals A Protein Kinase A-Dependent Mechanism by Which Rotavirus Affects the Distribution and mRNA Level of the Functional Tight Junction-Associated Protein, Occludin, in Human Differentiated Intestinal Caco-2 Cells

2007 ◽  
Vol 81 (16) ◽  
pp. 8579-8586 ◽  
Author(s):  
Isabelle Beau ◽  
Jacqueline Cotte-Laffitte ◽  
Raymonde Amsellem ◽  
Alain L. Servin
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Laser Scanning ◽  
Time Course ◽  
Mrna Level ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Infected Cells ◽  
Triton X ◽  
Kinase A

ABSTRACT We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

scholarly journals Protein Kinase A-Dependent Derepression of the Human Prodynorphin Gene via Differential Binding to an Intragenic Silencer Element

1998 ◽  
Vol 18 (12) ◽  
pp. 6921-6929 ◽  
Author(s):  
Angel M. Carrión ◽  
Britt Mellström ◽  
Jose R. Naranjo
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Time Course ◽  
Neuroblastoma Cells ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Human Neuroblastoma Cells ◽  
Basal Transcription ◽  
Human Neuroblastoma ◽  
Kinase A

ABSTRACT Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5′ deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5′ untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

scholarly journals Adrenergic Regulation and Diurnal Rhythm of p38 Mitogen-Activated Protein Kinase Phosphorylation in the Rat Pineal Gland

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 5194-5201 ◽  
Author(s):  
C. L. Chik ◽  
M. Mackova ◽  
D. Price ◽  
A. K. Ho
Keyword(s):  
Protein Kinase ◽  
Pineal Gland ◽  
Protein Kinase A ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Adrenergic Blocker ◽  
Rat Pineal Gland ◽  
Kinase A

Abstract In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both α- and β-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4β-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca2+/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a β-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca2+ signaling pathways are involved in NE regulation of p38MAPK.

scholarly journals Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations

2021 ◽  
Vol 118 (21) ◽  
pp. e2024716118
Author(s):  
Naeimeh Jafari ◽  
Jason Del Rio ◽  
Madoka Akimoto ◽  
Jung Ah Byun ◽  
Stephen Boulton ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Carney Complex ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Inhibitory Function ◽  
Wide Range ◽  
Liver Cancers ◽  
Kinase A

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Protein kinase A mediates novel serine-584 phosphorylation of HDAC4

2019 ◽  
Vol 97 (5) ◽  
pp. 526-535 ◽  
Author(s):  
Shanmukha K. Doddi ◽  
Githavani Kummari ◽  
Jagannadham M.V. ◽  
Arunasree M. Kalle
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Cell Line ◽  
Target Genes ◽  
K562 Cells ◽  
Pcr Analysis ◽  
Wild Type ◽  
Kinase A

Given the well-established diversified signaling pathways for histone deacetylase 4 (HDAC4) and the regulation of HDAC4 by several post-translational modifications (PTMs), including phosphorylation, sumoylation, and ubiquitination, an unbiased and detailed analysis of HDAC4 PTMs is needed. In this study, we used matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) to describe phosphorylation at serine 584 (Ser584) along with already-known dual phosphorylation at serines 265 and 266 (Ser265/266), that together regulate HDAC4 activity. Overexpression of site-specific HDAC4 mutants (S584A, S265/266A) in HEK 293T cells, followed by HDAC activity assays, revealed the mutants to be less active than the wild-type protein. In vitro kinase assays have established that Ser584 and Ser265/266 are phosphorylated by protein kinase A (PKA). Luciferase assays driven by the myocyte enhancer factor 2 (MEF2) promoter and real-time PCR analysis of the MEF2 target genes show that the S584A and S265/266A mutants are less repressive than the wild-type. Furthermore, treatment with PKA activators such as 8-Bromo-cAMP and forskolin, and silencing either by shRNA or its inhibitor H-89 in a mouse myoblast cell line (C2C12) and in a non-muscle human cell line (K562), confirmed in vivo phosphorylation of HDAC4 in C2C12 but not in K562 cells, indicating the specific functional significance of HDAC4 phosphorylation in muscle cells. Thus, we identified PKA-induced Ser584 phosphorylation of HDAC4 as a yet unknown regulatory mechanism of the HDAC4–MEF2 axis.

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