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- PDB-6wjf: PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar h... -

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Basic information

Entry
Database: PDB / ID: 6wjf
TitlePKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinoma
Components
  • DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
  • cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathway
KeywordsSIGNALING PROTEIN / fibrolamellar hepatoceullar carcinoma / PKA / cAMP / 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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsLu, 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
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 16, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
B: DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion
C: cAMP-dependent protein kinase type II-beta regulatory subunit
D: cAMP-dependent protein kinase type II-beta regulatory subunit


Theoretical massNumber of molelcules
Total (without water)187,0324
Polymers187,0324
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS, The structure fits the experimental SAXS data well with chi^2=1.2900.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9250 Å2
ΔGint-29 kcal/mol
Surface area69180 Å2

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Components

#1: Protein DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusion / DnaJ protein homolog 1 / Heat shock 40 kDa protein 1 / Heat shock protein 40 / Human DnaJ protein 1 ...DnaJ protein homolog 1 / Heat shock 40 kDa protein 1 / Heat shock protein 40 / Human DnaJ protein 1 / hDj-1 / PKA C-alpha


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 cAMP-dependent protein kinase type II-beta regulatory subunit / CAMP-dependent pathway


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

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1PKA RIIbeta holoenzyme with DnaJB1-PKAc fusion in fibrolamellar hepatoceullar carcinomaCOMPLEXall0RECOMBINANT
2DnaJ homolog subfamily B member 1,cAMP-dependent protein kinase catalytic subunit alpha fusionCOMPLEX#11RECOMBINANT
3cAMP-dependent protein kinase type II-beta regulatory subunitCAMP-dependent pathwayCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Rattus norvegicus (Norway rat)10116
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
Buffer solutionpH: 5.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11182 / Symmetry type: POINT

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