1. Biochemistry and Chemical Biology
  2. Cancer Biology
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An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma

Research Article
Cite this article as: eLife 2019;8:e44187 doi: 10.7554/eLife.44187
7 figures, 1 table, 1 data set and 3 additional files

Figures

Figure 1 with 3 supplements
Properties of the DNAJ-PKAc fusion enzyme.

(A) Structure of the DNAJ-PKAc fusion protein (PDB ID 4WB77). The DNAJ (orange) and PKAc domains (blue) are indicated. (B) Immunoblots of paired tumor and normal adjacent liver from FLC patients probed with antibodies to PKAc (top panels) and actin loading controls (bottom panels). DNAJ-PKAc (upper band) migrates with a slower mobility than the native C subunit in SDS-PAGE. (C–D) Immunofluorescence images of normal liver (left, C) and FLC (right, D) stained with antibodies against PKAc (green), RIIα (red) and DAPI (blue). Scale bar represents 50 μm. (E–F) Phosphoproteomic profiling of FLC. Statistical significance was calculated Significant differences in phosphopeptide expression between experiments were quantified with a two-tailed two sample t-test with unequal variances and Benjamini-Hochberg correction for multiple comparisons was applied (FDR ≤ 0.05), log2 ratio >1. (E) Volcano plot showing phosphosites upregulated (orange) and downregulated (black) in FLC as compared to normal adjacent liver. (F) Pie chart of putative kinase substrates (predicted by NetworKIN) increased in FLC. 82.8% of sites identified were in the NetworKIN platform. Percentages of sites ascribed to particular kinase are listed. ‘Other’ kinases include: CK1, TTK, GRK, RSK, MAK, JNK, ROCK, P70S6K, AMPK, CLK, HIPK2, PDHK, ACTR2, ATM, DMPK, IKK, MOK, NEK4, PKD1, PKG, TGFBR2, and p38-MAPK. (G) Schematic of DNAJ-PKAc in complex with heat shock protein 70 (Hsp70, red). (H) Immunoblot detection of Hsp70 in PKAc immune complexes from FLC and normal adjacent liver lysates (top). Loading controls indicate the levels of Hsp70 (middle) and both forms of PKA (bottom). (I–J) Proximity Ligation (PLA) detection of DNAJ-PKAc/Hsp70 complexes in (I) normal liver and J) FLC sections. Yellow puncta identify Hsp70-kinase sub-complexes, DAPI (blue) marks nuclei. Scale bar represents 20 μm. (K) Amalgamated data (PLA puncta/μm2) from eight normal (black) and 9 FLC (orange) sections. Data are shown as mean ±s.d., p<0.0001 by Student’s t-test (t = 10.98, df = 15).

https://doi.org/10.7554/eLife.44187.003
Figure 1—figure supplement 1
Altered PKA signaling in FLC.

(A) Immunoblots of paired normal adjacent liver and tumor from FLC patients probed with antibodies to RI (top) and RIIα (bottom). (B) Immunoblots of the same samples probed with an antibody recognizing phospho-PKA substrates (R-R-X-pS/pT). (C) RII overlay analysis to detect AKAPs in the same samples (D) List of select PKA substrates elevated in phosphoproteomic analysis of FLCs. Phosphorylated residues (red) and the scaffold protein KSR1(bold) are indicated.

https://doi.org/10.7554/eLife.44187.004
Figure 1—figure supplement 2
Kinase network rewiring in FLC.

Phospho-substrate analysis of phosphoproteomic data from FLCs showing a spectrum of kinases implicated in covalent modification of heterogeneous tumor tissue. Putative kinase substrates were classified using NetworKIN as described in Figure 1 and Materials adn methods. Bar graph depicts the percent change of kinase substrates elevated (orange) and attenuated (black) in FLC.

https://doi.org/10.7554/eLife.44187.005
Figure 1—figure supplement 3
Additional Proximity Ligation (PLA) detection of Hsp70 and PKAc in patient tissue.

(A and B) Full-size source images for Figure 1I & J, gray boxes indicate area shown. PLA signal between Hsp70 and PKAc (yellow) is less prominent in normal tissue (A) than in FLC (B). Dapi (blue) marks nuclei. (C and D) Images of the PLA signals in (A and B) after removing unfocused fluorescent signal with Keyence software haze reduction. (E and F) Control images for normal liver (E) and FLC (F) where PLA cannot occur due to omission of one secondary antibody. (G and H) Yellow channel signals of control normal liver (G) and FLC (H). Note greater background fluorescence in normal liver sections vs. FLC emanating from fat deposits. Scale bars are 50 µm. (I and J) Quantification of number of PLA puncta (I) and average puncta intensity (J). Data presented as mean ±s.d. with technical replicates as individual data points. Total areas quantified were. 349 mm2,. 392 mm2,. 131 mm2, and. 131 mm2, and total puncta measured were 2,943, 7,583, 733, and 47 for normal PLA, tumor PLA, normal control, and tumor control conditions, respectively.

https://doi.org/10.7554/eLife.44187.006
Figure 2 with 2 supplements
Generation and characterization of AML12DNAJ-PKAc cell lines.

(A) CRISPR-Cas9 gene editing of mouse chromosome eight in AML12 cells deleted a 400 kb region between intron 1 of the gene for Hsp40 (Dnajb1) and intron 1 of the gene for PKAc (Prkaca). (B) PCR detection of transcripts for the Gipc1, Ddx39 and Lphn1 genes encoded on the non-engineered strand of mouse chromosome 8. (C–E) Quantitative PCR detection of native mRNA transcripts in AML12 (black) and gene-edited (orange) cell lines. (C) Detection of native Dnajb1 mRNA transcripts, (D) Prkaca transcripts and (E) Dnajb1-Prkaca mRNA transcripts. Data (n = 3) is normalized to Gapdh (C–E) and relative to (C,D) wildtype AML12 or (E) clone 2. Error bars indicate mean ±s.d. (F) Amino acid sequence of the fusion protein DNAJ-PKAc is shown in orange and blue. Nucleotide sequence of the fusion gene from clone 14 AML12DNAJ-PKAc cells is shown below. (G) Immunoblot detection of both native and mutant PKAc in four clonal AML12DNAJ-PKAc cell lines. Top) DNAJ-PKAc fusion proteins (upper bands) and wildtype PKAc (lower bands) are indicated. The distribution of PKAc in wildtype AML12 cells, normal liver and FLC are included. Bottom) Actin loading control. (H) Immunoblot detection of PKA in Hsp70 immune complexes isolated from wildtype (AML12) and clone 14 AML12DNAJ-PKAc cells. Lysate loading controls indicate both forms of PKA (middle) and levels of Hsp70 (bottom). (I and J) Proximity Ligation (PLA) detection of proteins within 40–60 nm of each other in (I) AML12 and (J) AML12DNAJ-PKAc cells. Yellow puncta identify Hsp70-kinase sub-complexes. Actin stain (green) marks cytoskeleton and DAPI staining (blue) marks nuclei. (K) Box-whisker plots of Hsp70-kinase sub-complexes. Amalgamated data (PLA puncta/cell) from AML12 (black) and AML12DNAJ-PKAc (orange) cells. Number of cells analyzed over three independent experiments is indicated below each plot; data are shown as mean ±s.d., p<0.0001 by Student’s t-test (t = 14.16, df = 105).

https://doi.org/10.7554/eLife.44187.007
Figure 2—figure supplement 1
Additional characterization of AML12DNAJ-PKAc cells.

(A) Immunoblot detection of PKA RIα (top panel), RIIα (mid-top panel) and RIIβ (mid-lower panel) subunits in parental AML12 cells and four clonal AML12DNAJ-PKAc cell lines. Bottom) Actin loading control. (B) The specific activity (pmol/μg/min) of basal and cAMP responsive PKA activity in wildtype AML12 (black) and clone 14 AML12DNAJ-PKAc (orange) was measured by radioactive kinase assay using Kemptide as a substrate. PKI 5–24 inhibitor peptide specifically blocked PKA activity. Representative data (mean ±s.d.) from three independent experiments. Holm-Sidak T-tests were performed. (C–F) Motile properties of AML12DNAJ-PKAc cells as measured by scratch wound. Migration of (C) AML12 and (D) clone 14 AML12DNAJ-PKAc cells. Images were collected every 45 min over 24 hr of n > 3 replicates. Time 0 (t = 0) is immediately after scratch-wounding. Transmigration through matrigel of (E) AML12 and (F) AML12DNAJ-PKAc cells. Images were taken every 45 min for 48 hr of n > 3 replicates; representative data shown. Time 0 (t = 0) is immediately after scratch-wounding. Scale bar is 300 μm.

https://doi.org/10.7554/eLife.44187.008
Figure 2—figure supplement 2
Additional Proximity Ligation (PLA) detection of Hsp70 and PKAc in (A) AML12 and (B) AML12DNAJ-PKAc cells.

Yellow puncta identify Hsp70-kinase sub-complexes. Actin stain (green) marks cytoskeleton and DAPI staining (blue) marks nuclei. These data were used for the quantification of PKA-Hsp70 puncta in Figure 2K.

https://doi.org/10.7554/eLife.44187.009
Figure 3 with 1 supplement
Cell proliferation analyses and combination drug sensitivity screening of AML12DNAJ-PKAc cells.

(A) Cell growth of wildtype AML12 (black) and AML12DNAJ-PKAc (orange) cells measured by MTS colorimetric assay. Absorbance (AU) was measured over a time course of 72 hr. Data are expressed as mean ±s.d. (n = 3); p=0.01 (t = 4.49, df = 6). (B) In situ incorporation of BrdU as an independent means of assessing DNA synthesis. Representative panels of wildtype (left) AML12 and (right) AML12DNAJ-PKAc cells. Scale bar represents 50 μm. (C) Percentage of BrdU positive cells presented as mean ±s.d. (n = 3); p=0.0001 (t = 14.51, df = 4). (D) Clonogenic growth of (top) AML12 and (bottom) AML12DNAJ-PKAc cells. Cells were seeded at 200 cells/well in a 12 well plate and grown for two weeks in normal growth media followed by crystal violet staining. (E) Amalgamated data charting area of growth in each well is presented as box and whiskers plot (min-max; n = 3); p<0.0001 by Student’s t-test (t = 6.14, df = 17). (F) Dose-response curves monitor the cytotoxic effects of the Hsp70 inhibitor Ver-155008 alone in AML12 (black) and AML12DNAJ-PKAc (orange) cells. Cell viability was assessed by MTS. Concentrations of drug used in each condition are indicated below each column. (G and H) Scatterplots show relative resistance or sensitivity of (G) AML12 and (H) AML12DNAJ-PKAc cells to the combination of 125 different chemotherapeutic drugs with Ver-155008. Drug combinations in the lower right quadrant are more sensitive to drug treatment than those in the upper right quadrant. Three drug combinations (pink circles) were identified for further validation, as they were more toxic to cells expressing DNAJ-PKAc than cells only expressing wildtype kinase. (I) Heat map of a subset of these data compares AML12DNAJ-PKAc cell survival with and without Ver-155008. AML12DNAJ-PKAc cells show drug resistance when treated with binimetinib, cobimetinib, or trametinib alone (left, blue) but they are more sensitive when these drugs are combined with Ver-155008 (right, green). (J and K) Analysis of (J) wildtype AML12 and (K) AML12DNAJ-PKAc cell survival. Dose-response of cobimetinib alone, (gray) or in combination with Ver-155008 (pink). Drug concentrations (μM) are indicated.

https://doi.org/10.7554/eLife.44187.010
Figure 3—figure supplement 1
Repeat combination drug screens at lower concentrations (3 μM) of Ver-155008.

(A and B) Scatterplots show relative resistance or sensitivity of (A) AML12 and (B) AML12DNAJ-PKAc cells to the combination of 125 different chemotherapeutic drugs with Ver-155008. Drug combinations in the lower right quadrant are more sensitive to drug treatment than those in the upper right quadrant. Binimetinib, cobimetinib and trametinib are highlighted (pink circles). (C and D) Analysis of (C) wildtype AML12 and (D) AML12DNAJ-PKAc cell survival: Dose-response of the drug cobimetinib alone (gray) or in combination with Ver-155008 (pink). Drug concentrations (μM) are indicated.

https://doi.org/10.7554/eLife.44187.011
Heterogeneous activation of ERK signaling in FLCs.

(A) Immunoblots of paired tumor and normal adjacent liver from FLC patients probed with antibodies to phospho-ERK1/2 (top panel) and total ERK1/2 (bottom panel). (B) Immunofluorescence images of FLC section from patient #3 were stained with antibodies against phospho-ERK (yellow), total ERK (magenta) and DAPI (nuclei, blue). Scale bar represents 20 μm. (C) Enlarged region from (B) showing prominent phospho-ERK staining in a subset of tumor hepatocytes. (D) Salient ERK substrates identified in phosphoproteomic analysis of FLC. Gene names, degree of enrichment (log2difference tumor/normal) and primary phosphosite sequences (one letter code) are indicated. The protein kinase P90-RSK2 is highlighted. (E) Immunoblots of paired tumor and normal adjacent liver from FLC patients probed with antibodies to phospho-P90RSK (top panel). Actin loading control (bottom panel). (F) Immunofluorescence image of FLC section stained with antibodies against phospho-P90RSK (magenta), PKA RII (green) and the nuclear marker DAPI (blue). (G) Enlarged region from (F). Dashed lines) highlight increased nuclear accumulation of phospho-P90RSK signal. Scale bars indicate 20 μm.

https://doi.org/10.7554/eLife.44187.012
Figure 5 with 1 supplement
Pharmacologically targeting DNAJ-PKAc assemblies.

(A) Schematic of an AKAP-Lbc-KSR-1 macromolecular assembly that sequesters Hsp70 and DNAJ-PKAc with elements of the ERK kinase cascade. (B) Immunoblots of paired FLC and normal adjacent liver probed with antibodies to AKAP-Lbc (top panels) and PKAc (bottom panels). (C) Immunoblot detection of PKAc (top) in AKAP-Lbc immune complexes (upper-mid) from normal adjacent tissue and FLC. PKAc (lower-mid) and AKAP-Lbc (bottom) in tissue lysates are indicated. DNAJ-PKAc (red) is indicated. (D) Co-immunoprecipitation of signaling elements with AKAP-Lbc from AML12DNAJ-PKAc cells. Immunoblot detection of PKAc (top) and Hsp70 (upper-mid) in immune complexes isolated from AML12DNAJ-PKAc cells. PKAc (middle), Hsp70 (mid-lower) in lysates from wildtype and AML12DNAJ-PKAc cells. Actin (bottom) served as loading control. (E) Immunoblot detection of phospho-ERK1/2 (top) as an index of ERK kinase activity in cell lysates from AML12 and AML12DNAJ-PKAc cells. Bottom) Immunoblot detection of total ERK served as a loading control. Quantification of immunoblots (n = 4); mean ±s.d. and p=0.04 (t = 2.6, df = 6). (f–I) In situ immunofluorescence of basal ERK activity. Grayscale images depicting immunofluorescent detection of phospho-ERK1/2 in (F) wildtype and (H) AML12DNAJ-PKAc cells. Composite images of phospho-ERK1/2 (green), actin (red) and nuclei (blue) in (G) wildtype and (I) AML12DNAJ-PKAc cells. Scale bar represents 10 μm. (J) Immunoblot detection of phospho-ERK 1/2 in wildtype AML12 (lanes 1–4) and AML12DNAJ-PKAc cells (lanes 5–8). Cells were treated with 100 nM of the MEK inhibitor cobimetinib, 3 μM Ver-155008 or combination of both drugs. Bottom) Detection of total ERK served as loading control. (K) Clonogenic growth assay portraying crystal violet (blue) staining of AML12DNAJ-PKAc cell proliferation in the presence of cobimetinib (100 nM), Ver-155008 (3 µM) and both drugs in combination.

https://doi.org/10.7554/eLife.44187.013
Figure 5—figure supplement 1
Effect of combination treatment with trametinib and Ver-155008 on cell growth.

Clonogenic growth assay of AML12DNAJ-PKAc cells in the presence of the MEK inhibitor trametinib ± Hsp70 inhibition with Ver-155008.

https://doi.org/10.7554/eLife.44187.014
Interruption of the DNAJ-PKAc/Hsp70 interface reduces ERK activation: substrate bias towards ERK signaling in AML12DNAJ-PKAc cells.

(A) Schematics of native DNAJ-PKAc (left) and DNAJ-PKAc H33Q mutant that cannot bind Hsp70 (right, gray). (B) Mutation of the chaperonin-binding site (H33Q) on DNAJ-PKAc abrogates interaction with Hsp70. Endogenous HSP70 co-precipitates with DNAJ-PKAc in AML12 cells expressing FLAG-DNAJ-PKAc (lane 1), but not with FLAG-Hsp40 H33Q control (lane 2) or the FLAG-DNAJ-PKAc H33Q mutant (lane 3). (C) GFP-tagged AKAP-Lbc co-precipitates endogenous Hsp70 in AML12 cells expressing FLAG-DNAJ-PKAc (lane 2) but not in cells expressing the wildtype FLAG-PKAc (lane 1) or the FLAG-DNAJ-PKAc H33Q mutant (lane 3). (D) Immunoblot detection of phospho-ERK1/2 in AML12 cells transiently transfected with DNAJ-PKAc (lane 2) or DNAJ-PKAc H33Q (lane 3). Total ERK (middle) served as a loading control. Detection of PKAc (bottom) monitored transfection efficiency. Quantitation of blots from four experiments, p=0.01 (t = 3.406, df = 6) and p=0.03 (t = 2.758, df = 6). (E and F) Differential phosphoproteomic profiling of AML12DNAJ-PKAc cells. (E) Volcano plot showing abundance (orange) and reduction (black) of phosphopeptides in AML12DNAJ-PKAc cells. Statistical significance of biological replicates was calculated by Student’s t test with Log10-transformed p-values of individual phosphopeptides plotted against log2-transformed fold change; n = 6. (F) Pie chart of putative kinase substrates increased in AML12DNAJ-PKAc cells. Sites identified by NetworKIN platform. Individual kinases are listed. ‘Other’ kinases include: CK, ABL2, GRK, GSK3, JAK2, NLK, and SRC.

https://doi.org/10.7554/eLife.44187.015
Author response image 1

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyActinGreen-488Molecular probesR37110Manufacturer instructions
AntibodyActinRed-555Molecular probesR37112Manufacturer instructions
AntibodyAKAP-Lbc (VO96)Diviani et al., 2001rabbit polyclonal(1:1000)
AntibodyAmersham ECL Mouse IgG, HRP-linked F(ab')₂ fragment (from sheep)GE Life SciencesNA9310(1:10000)
AntibodyAmersham ECL Rabbit IgG, HRP-linked F(ab')₂ fragment (from donkey)GE Life SciencesNA9340(1:10000)
AntibodyActin betaSigma-AldrichA1978
mouse monoclonal
RRID:AB_476692
(1:2500)
AntibodyBrdUDakoM0744
mouse monoclonal
RRID:AB_10013660
(1:1000)
AntibodyDonkey anti-Mouse IgG, Alexa Fluor 555InvitrogenA-31570(1:500)
AntibodyDonkey anti-Mouse IgG, Alexa Fluor 488InvitrogenA-21202(1:800)
AntibodyDonkey anti-Rabbit IgG, Alexa Fluor 488InvitrogenR37118(1:500)
AntibodyDonkey anti-Rabbit
IgG, Alexa Fluor 555
InvitrogenA-31572(1:800)
AntibodyGAPDH-HRPNovusNB110-40405
mouse monoclonal
RRID:AB_669249
(1:1000)
AntibodyHsp70Proteintech10995–1
rabbit polyclonal
RRID:AB_2264230
WB (1:500), PLA in tissue (1:200), PLA in cells (1:500)
Antibodyp-44/42 ERKCST9102
rabbit polyclonal
RRID:AB_330744
(1:1000)
Antibodyp-44/42 ERKBD Transduction610123
mouse monoclonal
RRID:AB_397529
WB (1:1000), IHC (1:100)
Antibodyphospho-p44/42 MAPKCST9101
rabbit polyclonal
RRID:AB_331646
WB (1:500), IHC (1:100)
AntibodyPKAcBD Transduction610981
mouse monoclonal
RRID:AB_398294
WB (1:500), PLA in tissue (1:200), PLA in cells (1:500)
AntibodyPKAcCST5842
rabbit monoclonal
RRID:AB_10706172
IHC (1:500)
AntibodyRIaBD Transduction610610
mouse monoclonal
RRID:AB_397944
(1:1000)
AntibodyRIIaBD Transduction612243
mouse monoclonal
RRID:AB_399566
(1:1000)
AntibodyRIIbBD Transduction610626
mouse monoclonal
RRID:AB_397958
(1:1000)
Antibodyphospho-RSKThermo-FisherPA5-37829
rabbit polyclonal
RRID:AB_2554437
WB (1:500), IHC (1:100)
AntibodyFLAG M2 Magnetic BeadsSigma-AldrichM8823
mouse monoclonal
RRID:AB_2637089
IP (1:40)
AntibodyGFPRockland600-101-215
goat polyclonal
RRID:AB_218182
WB (1:1000), IP (1:700)
AntibodyRIBD Transduction610165
mouse monoclonal
RRID:AB_397566
(1:500)
Antibodyphospho-PKA substrates (RRXS*/T*)CST9624
rabbit monoclonal
RRID:AB_331817
(1:1000)
AntibodyNeutrAvidin-HRPThermo-Fisher31030(1:5000)
AntibodyRIIa and bMcCartney et al., 1995goat polyclonal(1:200)
Cell line (M. musculus)AML12ATCCATCC: CRL-2254
RRID:CVCL_0140
Obtained from KJR by way of Nelson Fausto lab (original ATCC depositor)
Chemical compound, drugDAPIThermo-Fisher62248Manufacturer instructions
Chemical compound, drugATP, [γ−32P]- 3000 Ci/mmol 10mCi/ml EasyTide, 100 µCiPerkin-ElmerBLU502A100UC
Chemical compound, drugBrdUInvitrogenB23151
Chemical compound, drugCobimetinibSigma-AldrichADV465749767
Chemical compound, drugTrametinibSigma-AldrichADV465749287
Chemical compound, drugDexamethasoneSigma-AldrichD4902
Chemical compound, drugDMEM/F-12Gibco11320033
Chemical compound, drugFetal Bovine SerumThermo-FisherA3382001
Chemical compound, drugGentamicin sulfate saltSigma-AldrichG1264
Chemical compound, drugITS Liquid Media SupplementSigma-AldrichI3146
Chemical compound, drugLipofectamine LTX with Plus ReagentThermo-Fisher15338100
Chemical compound, drugPuromycinSigma-AldrichP8833
Chemical compound, drugTransIT-LT1 Transfection ReagentMirusMIR2300
Chemical
compound, drug
Trypsin-EDTA (0.25%), phenol redGibco25200056
Chemical compound, drugCrystal VioletSigmaC3886
Chemical compound, drugVer-155008Sigma-Aldrich1134156-31-2
Commercial
assay or kit
CellTiter 96 AQueous One Solution Cell Proliferation AssayPromegaG3582
Commercial assay or kitCryoGrinder KirOPS DiagnosticsCG0801
Commercial assay or kitDuolink In Situ Orange Starter Kit Mouse/RabbitSigma-AldrichDUO92102
Commercial assay or kitGeneJET Genomic DNA purification kitThermoK0721
Commercial assay or kitPierce BCA Protein Assay KitThermo23225
Commercial assay or kitPowerUp SYBR Green Master MixThermo-FisherA25741
 Commercial assay or kitReverse Transcription SupermixBio-Rad1708840
Commercial assay or kitRNeasy Mini KitQiagen74106
Commercial assay or kitSignaTECT cAMP-Dependent Protein Kinase (PKA) Assay SystemPromegaV7480
Commercial assay or kitZero Blunt TOPO PCR Cloning KitThermo-Fisher450245
Peptide, recombinant proteinRII-biotinCarr et al., 1992
Peptide, recombinant proteinPKISigma-AldrichP7739
Recombinant DNA reagentDNAJ-PKAc FLAGThis paperIn-house modified pDEST12.2 (N-terminal FLAG)
Recombinant DNA reagentDNAJ-PKAc H33Q FLAGThis paperIn-house modified pDEST12.2 (N-terminal FLAG)
Recombinant DNA reagentDNAJB1 FLAGThis paperThis paperIn-house modified pDEST12.2 with N-terminal FLAG;
backbone from Invitrogen (discontinued)
Recombinant DNA reagentAKAP-Lbc GFPClonetech; Diviani et al., 2001pEGFP-N1 (Clontech) backbone
Recombinant DNA reagenthSpCas9-gDnajb1-Prkaca-2A-PuroThis paperRRID:Addgene_48138PX458 backbone; Dual U6-sgRNA cassettes
Sequenced-based reagentGipc1_FThis paperPCR primersGGGAAAGGACAAAAGGAACCC
Sequenced-based reagentGipc1_RThis paperPCR primersCAGGGCATTTGCACCCCATGCC
Sequenced-based reagentDdx39_FThis paperPCR primersCCGGGACTTTCTACTGAAGCC
Sequenced-based reagentDdx39_RThis paperPCR primersGAATGGCCTGGGGAATACAC
Sequenced-based reagentLphn1_FThis paperPCR primersACCCCTTCCAGATGGAGAATGT
Sequenced-based reagentLphn1_RThis paperPCR primersTGGGCAAGCATCTATGGCAC
Sequenced-based reagentDnajb1_ex2_FThis paperqPCR primersGGGACCAGACCTCGAACAAC
Sequenced-based reagentDnajb1_ex2_RThis paperqPCR primersGGCTAATCCTGGCTGGATAGAT
Sequenced-based reagentPrkaca_ex1_FThis paperqPCR primersAAGAAGGGCAGCGAGCAGGA
Sequenced-based reagentPrkaca_ex1_RThis paperqPCR primersGCCGGTGCCAAGGGTCTTGAT
Sequenced-based reagentGapdh_FThis paperqPCR primersATTTGGCCGTATTGGGCGCCT
Sequenced-based reagentGapdh_RThis paperqPCR primersCCCGGCCTTCTCCATGGTGG
Sequenced-based reagentDnaj-PKAc_FThis paperqPCR primersACGAGATCAAGCGAGCCTAC
Sequenced-based reagentDnaj-PKAc_RThis paperqPCR primersTTCCCACTCTCCTTGTGCTT
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)
Software, algorithmMaxQuant/Andromedahttps://www.maxquant.org/PMID: 19029910
Software,
algorithm
NetworKINhttp://networkin.info/PMID: 24874572
Software,
algorithm
Perseushttps://maxquant.net/perseus/PMID: 27348712
Software, algorithmPhosphoSitePlushttps://www.phosphosite.org

Data availability

Raw mass spectrometry data has been uploaded to MassIVE, an NIH supported MS data repository (MSV000083167).

The following data sets were generated
  1. 1
    MassIVE Repository
    1. M Golkowski
    2. RT Turnham
    3. SE Ong
    4. JD Scott
    (2017)
    An acquired scaffolding function of the DNAJ-PKAc fusion enhances oncogenesis in Fibrolamellar carcinoma.
    https://doi.org/10.25345/C5F01X

Additional files

Supplementary file 1

Combination drug screen data.

https://doi.org/10.7554/eLife.44187.016
Supplementary file 2

Phosphoproteomic data from FLCs and AML12DNAJ-PKAc cells.

https://doi.org/10.7554/eLife.44187.017
Transparent reporting form
https://doi.org/10.7554/eLife.44187.018

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