scholarly journals Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Cristina Olivieri ◽  
Caitlin Walker ◽  
Adak Karamafrooz ◽  
Yingjie Wang ◽  
V. S. Manu ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Substrate Binding ◽  
Chimeric Protein ◽  
Α Subunit ◽  
Primary Driver ◽  
Chimeric Construct ◽  
J Domain ◽  
Kinase A

AbstractAn aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

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 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.

Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A

2001 ◽  
Vol 79 (6) ◽  
pp. 471-480 ◽  
Author(s):  
Nathalie Thorin-Trescases ◽  
Sergei N Orlov ◽  
Sébastien Taurin ◽  
Nickolai O Dulin ◽  
Bruce G Allen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Protein Kinase A ◽  
Cholera Toxin ◽  
Antiproliferative Effect ◽  
Continuous Exposure ◽  
Α Subunit ◽  
Vsmc Proliferation ◽  
Kinase A

The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein αS subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G1 phase without induction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic α subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively). After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106. In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G1 phase.Key words: vascular smooth muscle, proliferation, cholera toxin, cAMP, protein kinase A.

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.

scholarly journals Functional Heterogeneity of Protein Kinase A Activation in Multipotent Stromal Cells

2020 ◽  
Vol 21 (12) ◽  
pp. 4442
Author(s):  
Pyotr A. Tyurin-Kuzmin ◽  
Maxim N. Karagyaur ◽  
Konstantin Yu. Kulebyakin ◽  
Daniyar T. Dyikanov ◽  
Vadim I. Chechekhin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Stromal Cells ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Multipotent Stromal Cells ◽  
Adenylyl Cyclases ◽  
Functional Heterogeneity ◽  
Pka Pathway ◽  
Kinase A

Multipotent stromal cells (MSC) demonstrate remarkable functional heterogeneity; however, its molecular mechanisms remain largely obscure. In this study, we explored MSC response to hormones, which activate Gs-protein / cyclic AMP (cAMP) / protein kinase A (PKA) dependent signaling, at the single cell level using genetically encoded biosensor PKA-Spark. For the first time, we demonstrated that about half of cultured MSCs are not able to activate the cAMP/PKA pathway, possibly due to the limited availability of adenylyl cyclases. Using this approach, we showed that MSC subpopulations responding to various hormones largely overlapped, and the share of responding cells did not exceed 40%. Using clonal analysis, we showed that signaling heterogeneity of MSC could be formed de novo within 2 weeks.

scholarly journals The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02880-20
Author(s):  
E. Keats Shwab ◽  
Praveen R. Juvvadi ◽  
Greg Waitt ◽  
Shareef Shaheen ◽  
John Allen ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Protein Kinase ◽  
Aspergillus Fumigatus ◽  
Protein Kinase A ◽  
Molecular Mechanisms ◽  
Targeted Mutagenesis ◽  
Human Pathogen ◽  
Content Type ◽  
Kinase A ◽  
Microbial Disease

ABSTRACTProtein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

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