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Yorodumi- PDB-6wjg: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h... -
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-Basic information
Entry | Database: PDB / ID: 6wjg | ||||||||||||
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Title | PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma | ||||||||||||
Components |
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Keywords | SIGNALING PROTEIN / fibrolamellar hepatoceullar carcinoma / PKA / Kinase | ||||||||||||
Function / homology | Function and homology information GPER1 signaling / PKA activation in glucagon signalling / CREB1 phosphorylation through the activation of Adenylate Cyclase / DARPP-32 events / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 ...GPER1 signaling / PKA activation in glucagon signalling / CREB1 phosphorylation through the activation of Adenylate Cyclase / DARPP-32 events / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Factors involved in megakaryocyte development and platelet production / Regulation of PLK1 Activity at G2/M Transition / Hedgehog 'off' state / PKA activation / response to antipsychotic drug / PKA-mediated phosphorylation of CREB / PKA-mediated phosphorylation of key metabolic factors / mitochondrial protein catabolic process / sperm head / ROBO receptors bind AKAP5 / negative regulation of inclusion body assembly / HDL assembly / Regulation of glycolysis by fructose 2,6-bisphosphate metabolism / cAMP-dependent protein kinase regulator activity / regulation of protein binding / nucleotide-activated protein kinase complex / high-density lipoprotein particle assembly / Vasopressin regulates renal water homeostasis via Aquaporins / Rap1 signalling / renal water homeostasis / regulation of protein processing / positive regulation of ATP-dependent activity / transcription regulator inhibitor activity / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cell communication by electrical coupling involved in cardiac conduction / cAMP-dependent protein kinase inhibitor activity / cAMP-dependent protein kinase / cellular response to cold / Loss of phosphorylation of MECP2 at T308 / sperm capacitation / regulation of osteoblast differentiation / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / cAMP-dependent protein kinase activity / ciliary base / cAMP-dependent protein kinase complex / negative regulation of glycolytic process through fructose-6-phosphate / AMP-activated protein kinase activity / postsynaptic modulation of chemical synaptic transmission / cellular response to glucagon stimulus / Triglyceride catabolism / protein kinase A regulatory subunit binding / ATPase activator activity / protein kinase A catalytic subunit binding / plasma membrane raft / chaperone cofactor-dependent protein refolding / PKA activation in glucagon signalling / : / Regulation of MECP2 expression and activity / mesoderm formation / HSF1-dependent transactivation / regulation of cardiac conduction / RET signaling / DARPP-32 events / response to unfolded protein / Interleukin-3, Interleukin-5 and GM-CSF signaling / sperm flagellum / Regulation of HSF1-mediated heat shock response / Mitochondrial protein degradation / regulation of macroautophagy / regulation of cardiac muscle contraction / Attenuation phase / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Hedgehog 'off' state / negative regulation of smoothened signaling pathway / regulation of cellular response to heat / regulation of proteasomal protein catabolic process / cAMP binding / protein folding chaperone / positive regulation of gluconeogenesis / forebrain development / Ion homeostasis / regulation of ryanodine-sensitive calcium-release channel activity / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / sperm midpiece / negative regulation of TORC1 signaling / protein kinase A signaling / Hsp70 protein binding / Recruitment of NuMA to mitotic centrosomes / cellular response to epinephrine stimulus / calcium channel complex / Anchoring of the basal body to the plasma membrane / regulation of cytosolic calcium ion concentration / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / protein export from nucleus / protein serine/threonine/tyrosine kinase activity Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) Rattus norvegicus (Norway rat) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.2 Å | ||||||||||||
Authors | Lu, T.-W. / Aoto, P.C. / Weng, J.-H. / Nielsen, C. / Cash, J.N. / Hall, J. / Zhang, P. / Simon, S.M. / Cianfrocco, M.A. / Taylor, S.S. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: PLoS Biol / Year: 2020 Title: Structural analyses of the PKA RIIβ holoenzyme containing the oncogenic DnaJB1-PKAc fusion protein reveal protomer asymmetry and fusion-induced allosteric perturbations in fibrolamellar hepatocellular carcinoma. Authors: Tsan-Wen Lu / Phillip C Aoto / Jui-Hung Weng / Cole Nielsen / Jennifer N Cash / James Hall / Ping Zhang / Sanford M Simon / Michael A Cianfrocco / Susan S Taylor / Abstract: When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the ...When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC. | ||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6wjg.cif.gz | 331.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6wjg.ent.gz | 268.9 KB | Display | PDB format |
PDBx/mmJSON format | 6wjg.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wj/6wjg ftp://data.pdbj.org/pub/pdb/validation_reports/wj/6wjg | HTTPS FTP |
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-Related structure data
Related structure data | 21693MC 6wjfC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 47337.984 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: DNAJB1, DNAJ1, HDJ1, HSPF1, PRKACA, PKACA / Production host: Escherichia coli (E. coli) References: UniProt: P25685, UniProt: P17612, cAMP-dependent protein kinase #2: Protein | Mass: 46177.852 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Prkar2b / Production host: Escherichia coli (E. coli) / References: UniProt: P12369 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (natural) |
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Source (recombinant) |
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Buffer solution | pH: 5.8 | ||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELDBright-field microscopy |
Image recording | Electron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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Symmetry | Point symmetry: C2 (2 fold cyclic) |
3D reconstruction | Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69605 / Symmetry type: POINT |