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- EMDB-21692: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h... -

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Basic information

Entry
Database: EMDB / ID: EMD-21692
TitlePKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma
Map dataC1-symmetry map
Sample
  • Complex: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma
    • Complex: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
      • Protein or peptide: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
    • Complex: cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathway
      • Protein or peptide: cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathway
Keywordsfibrolamellar hepatoceullar carcinoma / PKA / cAMP / Kinase / SIGNALING PROTEIN
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 / sperm head / ROBO receptors bind AKAP5 / negative regulation of inclusion body assembly / HDL assembly / high-density lipoprotein particle assembly / Regulation of glycolysis by fructose 2,6-bisphosphate metabolism / cAMP-dependent protein kinase regulator activity / regulation of protein binding / Vasopressin regulates renal water homeostasis via Aquaporins / Rap1 signalling / nucleotide-activated protein kinase complex / positive regulation of ATP-dependent activity / renal water homeostasis / regulation of protein processing / transcription regulator inhibitor activity / protein localization to lipid droplet / regulation of bicellular tight junction assembly / cellular response to parathyroid hormone stimulus / cAMP-dependent protein kinase inhibitor activity / cell communication by electrical coupling involved in cardiac conduction / 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 / negative regulation of glycolytic process through fructose-6-phosphate / ciliary base / cAMP-dependent protein kinase complex / 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 / regulation of cardiac conduction / HSF1-dependent transactivation / RET signaling / DARPP-32 events / response to unfolded protein / Interleukin-3, Interleukin-5 and GM-CSF signaling / Regulation of HSF1-mediated heat shock response / sperm flagellum / 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 / regulation of proteasomal protein catabolic process / negative regulation of smoothened signaling pathway / regulation of cellular response to heat / protein folding chaperone / protein kinase A signaling / forebrain development / cAMP binding / positive regulation of gluconeogenesis / regulation of ryanodine-sensitive calcium-release channel activity / Ion homeostasis / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / negative regulation of TORC1 signaling / Recruitment of mitotic centrosome proteins and complexes / sperm midpiece / Hsp70 protein binding / Recruitment of NuMA to mitotic centrosomes / cellular response to epinephrine stimulus / calcium channel complex / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / regulation of cytosolic calcium ion concentration / regulation of heart rate / protein export from nucleus / protein serine/threonine/tyrosine kinase activity / CD209 (DC-SIGN) signaling
Similarity search - Function
cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / HSP40/DnaJ peptide-binding / Chaperone DnaJ, C-terminal / DnaJ C terminal domain / Nt-dnaJ domain signature. / DnaJ domain, conserved site / DnaJ domain ...cAMP-dependent protein kinase regulatory subunit / cAMP-dependent protein kinase regulatory subunit, dimerization-anchoring domain / Regulatory subunit of type II PKA R-subunit / RIIalpha, Regulatory subunit portion of type II PKA R-subunit / HSP40/DnaJ peptide-binding / Chaperone DnaJ, C-terminal / DnaJ C terminal domain / Nt-dnaJ domain signature. / DnaJ domain, conserved site / DnaJ domain / DnaJ molecular chaperone homology domain / dnaJ domain profile. / Chaperone J-domain superfamily / DnaJ domain / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / cAMP-dependent protein kinase catalytic subunit / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Extension to Ser/Thr-type protein kinases / AGC-kinase, C-terminal / AGC-kinase C-terminal domain profile. / RmlC-like jelly roll fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
cAMP-dependent protein kinase type II-beta regulatory subunit / cAMP-dependent protein kinase catalytic subunit alpha / DnaJ homolog subfamily B member 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsLu T-W / Aoto PC
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM130389 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM34921 United States
National Institutes of Health/Office of the DirectorS10OD020011 United States
CitationJournal: 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
DepositionApr 13, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6wjf
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21692.png

Downloads & links

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Map

FileDownload / File: emd_21692.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC1-symmetry map
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.10794525 - 0.17533486
Average (Standard dev.)-0.0002850975 (±0.004777017)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1080.175-0.000

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Supplemental data

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Sample components

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Entire : PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h...

EntireName: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma
Components
  • Complex: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma
    • Complex: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
      • Protein or peptide: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
    • Complex: cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathway
      • Protein or peptide: cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathway

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Supramolecule #1: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h...

SupramoleculeName: 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...

SupramoleculeName: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: cAMP-dependent protein kinase type II-beta regulatory subunit

SupramoleculeName: cAMP-dependent protein kinase type II-beta regulatory subunit
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Rattus norvegicus (Norway rat)

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Macromolecule #1: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase c...

MacromoleculeName: 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: cAMP-dependent protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.337984 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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, cAMP-dependent protein kinase catalytic subunit alpha

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Macromolecule #2: cAMP-dependent protein kinase type II-beta regulatory subunit

MacromoleculeName: cAMP-dependent protein kinase type II-beta regulatory subunit
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 46.177852 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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: cAMP-dependent protein kinase type II-beta regulatory subunit

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 5.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm 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 modelType of model: OTHER / Details: Stochastic gradient descent
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11182

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