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- EMDB-21692: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-21692 | ||||||||||||
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Title | PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma | ||||||||||||
![]() | C1-symmetry map | ||||||||||||
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![]() | fibrolamellar hepatoceullar carcinoma / PKA / ![]() ![]() ![]() | ||||||||||||
Function / homology | ![]() 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 / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | ||||||||||||
Method | ![]() ![]() | ||||||||||||
![]() | Lu T-W / Aoto PC | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 40.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.1 KB 13.1 KB | Display Display | ![]() |
Images | ![]() | 40.3 KB | ||
Filedesc metadata | ![]() | 5.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6wjfMC ![]() 6wjgC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | C1-symmetry map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h...
Entire | Name: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma |
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Components |
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-Supramolecule #1: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h...
Supramolecule | Name: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase c...
Supramolecule | Name: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: cAMP-dependent protein kinase type II-beta regulatory subunit
Supramolecule | Name: cAMP-dependent protein kinase type II-beta regulatory subunit type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() ![]() ![]() |
-Macromolecule #1: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase c...
Macromolecule | Name: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ![]() |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 47.337984 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: GKDYYQTLGL ARGASDEEIK RAYRRQALRY HPDKNKEPGA EEKFKEIAEA YDVLSDPRKR EIFDRYGEEV KEFLAKAKED FLKKWESPA QNTAHLDQFE RIKTLGTGSF GRVMLVKHKE TGNHYAMKIL DKQKVVKLKQ IEHTLNEKRI LQAVNFPFLV K LEFSFKDN ...String: GKDYYQTLGL ARGASDEEIK RAYRRQALRY HPDKNKEPGA EEKFKEIAEA YDVLSDPRKR EIFDRYGEEV KEFLAKAKED FLKKWESPA QNTAHLDQFE RIKTLGTGSF GRVMLVKHKE TGNHYAMKIL DKQKVVKLKQ IEHTLNEKRI LQAVNFPFLV K LEFSFKDN SNLYMVMEYV PGGEMFSHLR RIGRFSEPHA RFYAAQIVLT FEYLHSLDLI YRDLKPENLL IDQQGYIQVT DF GFAKRVK GRTWTLCGTP EYLAPEIILS KGYNKGVDWW ALGVLIYEMA AGYPPFFADQ PIQIYEKIVS GKVRFPSHFS SDL KDLLRN LLQVDLTKRF GNLKNGVNDI KNHKWFATTD WIAIYQRKVE APFIPKFKGP GDTSNFDDYE EEEIRVSINE KCGK EFSEF UniProtKB: DnaJ homolog subfamily B member 1, ![]() |
-Macromolecule #2: cAMP-dependent protein kinase type II-beta regulatory subunit
Macromolecule | Name: cAMP-dependent protein kinase type II-beta regulatory subunit type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 46.177852 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MSIEIPAGLT ELLQGFTVEV LRHQPADLLE FALQHFTRLQ QENERKGAAR FGHEGRTWGD AGAAAGGGTP SKGVNFAEEP MRSDSENGE EEEAAEAGAF NAPVINRFTR RASVCAEAYN PDEEEDDAES RIIHPKTDDQ RNRLQEACKD ILLFKNLDPE Q MSQVLDAM ...String: MSIEIPAGLT ELLQGFTVEV LRHQPADLLE FALQHFTRLQ QENERKGAAR FGHEGRTWGD AGAAAGGGTP SKGVNFAEEP MRSDSENGE EEEAAEAGAF NAPVINRFTR RASVCAEAYN PDEEEDDAES RIIHPKTDDQ RNRLQEACKD ILLFKNLDPE Q MSQVLDAM FEKLVKEGEH VIDQGDDGDN FYVIDRGTFD IYVKCDGVGR CVGNYDNRGS FGELALMYNT PRAATITATS PG ALWGLDR VTFRRIIVKN NAKKRKMYES FIESLPFLKS LEVSERLKVV DVIGTKVYND GEQIIAQGDS ADSFFIVESG EVR ITMKRK GKSDIEENGA VEIARCLRGQ YFGELALVTN KPRAASAHAI GTVKCLAMDV QAFERLLGPC MEIMKRNIAT YEEQ LVALF GTNMDIVEPT A UniProtKB: ![]() |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Buffer | pH: 5.8 |
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Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 80.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: OTHER / Details: Stochastic gradient descent |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11182 |