1. Cancer Biology
  2. Cell Biology
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One reporter for in-cell activity profiling of majority of protein kinase oncogenes

  1. Iva Gudernova
  2. Silvie Foldynova-Trantirkova
  3. Barbora El Ghannamova
  4. Bohumil Fafilek
  5. Miroslav Varecha
  6. Lukas Balek
  7. Eva Hruba
  8. Lucie Jonatova
  9. Iva Jelinkova
  10. Michaela Kunova Bosakova
  11. Lukas Trantirek
  12. Jiri Mayer
  13. Pavel Krejci  Is a corresponding author
  1. Masaryk University, Czech Republic
  2. Central European Institute of Technology, Masaryk University, Czech Republic
  3. St. Anne's University Hospital, Czech Republic
  4. Hematology and Oncology, Masaryk University Hospital, Czech Republic
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Cite this article as: eLife 2017;6:e21536 doi: 10.7554/eLife.21536
8 figures and 3 additional files

Figures

Figure 1 with 8 supplements
Development of luciferase and fluorescent reporters based on a human EGR1 promoter.

(A) The activity of various reporters, based on promoters of FGF2-responsive genes, cloned into a pGL4.17 vector expressing firefly luciferase. The FGF2-mediated trans-activation (fold-change compared to unstimulated cells) of these reporters in RCS cells was determined by the dual-luciferase assay. Insert, induction of EGR1 protein expression in RCS cells treated with FGF2. (BE) Four consecutive rounds of EGR1 promoter sequence optimization leading to the pKrox24(2xD-E_inD)Luc reporter, including 5’-prime shortening (B,C), 3’-prime shortening (D), and addition of repetitive D-elements (E) to the originally cloned EGR1 promoter (vectors outlined in Figure 1—figure supplement 2). The presented data were generated through dual-luciferase assays in RCS cells, with ‘n’ describing the number of independent experiments. Statistically significant differences are highlighted (Student´s t-test; **p<0.01, ***p<0.001). (F, G) FGF2-mediated induction of dTomato protein expression (F) and fluorescence (G) in RCS cells transiently transfected with pKrox24(2xD-E)dTomato or pKrox24(MapErk)dTomato reporters. Bar, 150 µm. (H) Transactivation of pKrox24(2xD-E)dTomato in RCS cells induced by forced expression of the constitutively active FGFR3 K650M mutant, determined by live cell imaging of dTomato fluorescence over 24 hr. The dTomato induction was suppressed by the FGFR inhibitor AZD1480. (I) Immunoblot validation of DsRed induction and ERK phosphorylation (p) in RCS cells transfected with FGFR3 K650M mutant together with pKrox24(2xD-E)DsRed for 16 hr. Actin and total ERK levels served as loading controls.

https://doi.org/10.7554/eLife.21536.002
Figure 1—figure supplement 1
FGF2 induces EGR1 expression dependent on ERK MAP kinase.

(A) Immunoblot analysis of EGR1 protein induction in cultured cells treated with FGFR ligand FGF2 (40 ng/ml in hESC, hiPSC and NIH3T3; 10 ng/ml +1 µg/ml heparin in 293T, LP1 and KMS11 cells) for indicated times. hESC, human embryonic stem cells; hiPSC, human induced pluripotent stem cells; NIH3T3, mouse embryonal fibroblasts; 293T, human embryonal kidney cells; LP1, KMS11 human multiple myeloma cell lines. Actin serves as the loading control. (B) RCS cells were treated with ERK MAP kinase pathway inhibitor PD0325901 for 30 min prior FGF2 addition, and analyzed for ERK activating phosphorylation (pERK) and EGR1 induction one hour later. EGR1 induction by FGF2 depends on ERK activity. Actin and total ERK levels serve as loading controls. Data show representative experiment for three independent experiments.

https://doi.org/10.7554/eLife.21536.003
Figure 1—figure supplement 2
Schematic outline of human EGR1 promoter sequences cloned into the promoterless pGL4.17 vector carrying firefly luciferase, and analyzed for FGF2-mediated trans-activation as shown by Figure 1A–E.

The variants generated during four rounds of experimental optimization are indicated. TSS, transcription start site.

https://doi.org/10.7554/eLife.21536.004
Figure 1—figure supplement 3
Analysis workflow of the dual-luciferase assay.

Cells were transfected with promoterless pGL4.17 vector expressing firefly luciferase or containing cloned EGR1 promoter (pGL4.17-EGR1-D), together with vector carrying Renilla luciferase under constant promoter (pRL-TK) (transfection control). Chemiluminescence signals generated by each luciferase were determined by dual-luciferase assay (left graph; n = 8, four biological replicates each measured twice) and used to calculate F-luc/R-luc ratios for control and FGF2-treated cells (middle graph). These ratios were used to calculate a fold difference in pGL4.17-EGR1-D trans-activation between untreated and FGF2-stimulated cells (right graph).

https://doi.org/10.7554/eLife.21536.005
Figure 1—figure supplement 4
The extent of pKrox24(2xD-E_inD)Luc reporter trans-activation with increasing FGF2 concentrations in RCS cells.

Data are generated by dual-luciferase assay and represent an average from indicated number of independent experiments (n) with SEM.

https://doi.org/10.7554/eLife.21536.006
Figure 1—figure supplement 5
Validation of pKrox24(2xD-E_inD)Luc reporter in cellular models to FGFR signaling.

(A) Cultured 293T and NIH3T3 cells were transfected with pKrox24(2xD-E_inD)Luc, treated with FGF2 24 hr later and analyzed by luciferase assay after 20 hr. FGF2 trans-activated pKrox24(2xD-E_inD)Luc in both cell types. (B) hESC, hiPSC and KMS11 cells were transfected similar to (A) and endogenous high levels of FGF signaling were inhibited by small chemical inhibitors of FGFR catalytic activity AZD1480 (2 µM) and BGJ398 (10, 20, 40, 100 nM). Statistically significant differences are indicated (Student´s t-test; *p<0.05, **p<0.01, ***p<0.001). n, number of independent experiments.

https://doi.org/10.7554/eLife.21536.007
Figure 1—figure supplement 6
Generation of pKrox24(MapErk) reporters.

Conserved transcription factor binding sites were mapped to human EGR1 promoter together with information obtained during development of pKrox24(2xD-E_inD) reporters. A region crucial for the activity of the reporter overlapped with evolutionary conserved element (yellow box) and thus it was analyzed for transcription factors binding sites. Selected transcription factor responsive elements were then used for construction of the synthetic reporter where binding sites (BS) for SP1, CREB1, ELK1, ELK4, SRF and EGR1 itself were cloned five times in a row into pGL4.26 vector. TSS, transcription start site.

https://doi.org/10.7554/eLife.21536.008
Figure 1—figure supplement 7
Comparison of transactivation capacity and basal activity of pKrox24(MapErk)and pKrox24(2xD-E_inD) reporters.

(A) Comparison of FGF2-mediated trans-activation pKrox24(2xD-E_inD)Luc, pKrox24(MapErk)Luc and pGL4.17-EGR1-D reporters in RCS cells. Statistically significant differences are indicated (Student´s t-test; *p<0.05, **p<0.01). (B) Determination of basal levels of pKrox24(2xD-E_inD)Luc and pKrox24(MapErk)Luc activity in untreated 293T and RCS cells, transfected with indicated vectors for 24 hr, parental vectors are also shown. Data generated by dual-luciferase assay. n, number of independent experiments.

https://doi.org/10.7554/eLife.21536.009
Figure 1—figure supplement 8
FGF-mediated transactivation of constructs containing D-E or MapErk promoter elements combined with dTomato or DsRed reporters.

(A) RCS cells were transfected with pCLuc-Basic2 vector expressing dTomato under the pKrox24(2xD-E) promoter and treated with FGF2 (50 ng/ml) for 24 hr. dTomato expression was monitored by automatic incubation microscope BioStation CT (Nikon) over the entire 24 hr period of time and plotted (B) against signal recorded in FGF2-naïve cells transfected with pKrox24(2xD-E)dTomato. Scale bar 150 µm. (C) RCS cells were transiently transfected with activating FGFR3 mutant K650M together with pKrox24(2xD-E)dTomato vector for 24 hr alone or in the presence of FGFR inhibitor AZD1480. Representative images at 24 hr were acquired using confocal microscope Carl Zeiss LSM 700. Bar, 150 µm. dTomato fluorescence was recorded by BioStation CT over the entire 24 hr period of time, data are graphed and presented at Figure 1H. (D) Immunoblot analysis of dTomato and DsRed induction in FGF2-treated RCS cells transfected with vectors expressing DsRed and dTomato under the control of both pKrox24(2xD-E) and pKrox24(MapErk) promoters. Controls are non-transfected cells or those transfected with empty plasmid. Actin serves as the loading control. Numbers of independent experiments (n): 4 (A), 4 (B), 3 (C), 3 (D).

https://doi.org/10.7554/eLife.21536.010
RTK cloning and validation (part 1).

Full-length human RTK cDNA was cloned into pcDNA3.1 vectors and equipped with a C-terminal V5/His epitope. Mutants were created by site-directed mutagenesis. The RTKs were expressed in 293T cells, and their activation was probed by immunoblot with antibodies that recognize the given RTK only when it is phosphorylated (p) at a specific motif, with exception of phosphorylated DDR1 and DDR2 which were detected with pan-pY antibody. A total of 37 wild-type (WT) RTKs and 241 of their mutants were obtained, including disease-associated loss-of-function and gain-of-function mutants, and experimental kinase-inactive mutants (KD). Treatment with the cognate ligands of DDR1, DDR2, KIT, and VEGFR2 was used for the activation of these RTKs.

https://doi.org/10.7554/eLife.21536.011
RTK cloning and validation (part 2).
https://doi.org/10.7554/eLife.21536.012
Figure 4 with 1 supplement
RTKs induce EGR1 protein expression.

(A, B) Immunoblot analyses of EGR1 induction in 293T cells transfected with wild-type (WT) or mutated RTKs for 24 hr. Cells transfected with empty plasmids serve as the transfection control, and actin serves as the loading control. (A) Green, RTK induces EGR1; red, no EGR1 induction by the RTK; * RTKs that induced EGR1 but were not autophosphorylated (Figures 2 and 3); RTKs that were autophosphorylated but did not induce EGR1; L RTKs activated by the addition of their cognate ligands.

https://doi.org/10.7554/eLife.21536.013
Figure 4—figure supplement 1
EGR1 expression induced by non-receptor tyrosine kinases, serine/threonine kinases C-RAF and B-RAF, and RAS small GTPase.

(A) Immunoblot validation of cytoplasmic tyrosine kinases (ABL, BCR-ABL, ITK, SYK, TEC, TYK2, ZAP70, BLK, FGR, FYN, YES, LCK, LYN), serine/threonine kinases (B-RAF, C-RAF) and small GTPase RAS expression and activation in 293T cells. Cells transfected with empty plasmid serve as transfection control. Actin serves as the loading control. Control, untransfected cells. WT, wild-type RAS, V12, active RAS mutant. Two variants (p190 and p210) of oncogenic BCR-ABL are shown. CAAX, active C-RAF variant; V600E, oncogenic B-RAF mutant. (B) Immunoblot analysis of EGR1 protein induction in 293T cells, following 24 hr of expression of the indicated proteins in 293T cells. (C) Compilation of the data generated in (B). Green, expression of given protein induces EGR1; red, no EGR1 protein induction; * kinases that induced EGR1 but were not found autophosphorylated; kinases which did not induce EGR1 but were found autophosphorylated. Data show a representative experiment for three independent experiments.

https://doi.org/10.7554/eLife.21536.014
Figure 5 with 1 supplement
In-cell RTK activity profiling with BCR-ABL and EGFR inhibitors.

(A) Activity of BCR-ABL inhibitors ponatinib (Pona.), imatinib (Ima.), dasatinib (Dasa.), bosutinib (Bosu.), and nilotinib (Nilo.) against 28 wild-type RTKs, evaluated in 293T cells transfected with RTKs and treated with inhibitors for 20–24 hr. The panel compiles data from immunoblot detections of activated RTKs, each treated with inhibitor concentrations derived from the experiments shown in Figure 5—figure supplement 1. Only one concentration is shown for nilotinib due to its cell toxicity at higher concentrations. Asterisks highlight the previously unreported nilotinib targets LTK and INSR (Supplementary file 1E; Figure 5—figure supplement 1). (B) Activity profiling of 30 wild-type (wt) RTKs and 116 of their active mutants in the presence of 0.5 µM osimertinib. 293T cells were transfected with RTK vectors together with pKrox24(2xD-E_inD)Luc24 hr before osimertinib treatment (for 24 hr). The colors reflect the osimertinib-mediated inhibition of pKrox24(2xD-E_inD)Luctrans-activation induced by a given RTK, relative to cells untreated with osimertinib. Basal levels of osimetrinib-mediated inhibition of pKrox24(2xD-E_inD)Luc were obtained from cells transfected with empty plasmid and then subtracted from the data. (C) 293T cells were transfected with wt LTK or its mutants, and treated with osimertinib (Osi.) for 24 hr. The LTK autophosphorylation (p) reflect LTK activity. Total LTK and actin serve as loading controls. (D) Cell-free kinase assays were carried out with recombinant LTK or EGFR and osimertinib added to the kinase reaction. Phosphorylation (p) of a recombinant STAT1 and autophosphorylation was used to detect LTK and EGFR activation, respectively. Samples with omitted ATP serve as negative controls for kinase activity.

https://doi.org/10.7554/eLife.21536.015
Figure 5—figure supplement 1
Analyses of cytotoxicity and kinase activities of BCR-ABL and EGFR inhibitors.

(A) 293T cells were treated with ponatinib, imatinib, dasatinib, bosutinib, nilotinib and osimertinib for 24 hr and the cell amounts were determined by cell counting. Data represent average from three independent experiments (two biological replicates for each point in every experiment) with indicated SD. 0, cells treated with TKI vehicle DMSO. With exception of dasatinib, the inhibitors did not affect 293T proliferation at the concentration ranges (red lines) used in RTK screening (Figure 5A). (B, C) Cells were transfected with human wild-type ABL, and p190 and p210 variants of BCR-ABL, and wild-type human EGFR or its activating mutant T790M, treated with inhibitors for 24 hr and analyzed for activating phosphorylation (p) by immunoblot. Actin and total levels of expressed kinases serve as loading controls. Cells transfected with empty plasmid serve as transfection control. (D) Evidence demonstrating that 2 and 5 μM nilotinib inhibits expression of transfected FGFR2, MET and RON. (E) Inhibition of LTK and INSR autophosphorylation by nilotinib. (BE) Data show a representative experiment for three independent experiments.

https://doi.org/10.7554/eLife.21536.016
Author response image 1

(A) 293T cells were transfected with pKrox24 reporters together with empty vector and vectors expressing wild-type (WT) FGFR3 or its activating mutants G380R (associated with achondroplasia) and Y373C (associated with thanatophoric dysplasia). Note the differences in pKrox24 transactivation (upper graphs), and EGR1 induction (lower blot), which correspond to well documented differences in relative activity of the FGFR3 variants (see, for instance Krejci et al., PLoS ONE. 2008; 3: e3961), which is as follows: wt FGFR3<G380R<Y373C. K508M; kinase-dead FGFR3 mutant. (B) 293T cells were transfected with pKrox24 reporters together with empty vector and vectors expressing RTKs known to activate ERK pathway either weakly (INSR, IGF1R) or strongly (FGFR1, TRKB). Note the differences in pKrox24 transactivation (upper graphs), and EGR1 induction (lower blot), between strong and weak ERK activating RTKs. (Graphs) Values are averages from four biological replicates (each measured twice) with indicated S.D. Statistically significant differences are highlighted (Student's t-test; ***p<0.001). The levels of RTK expression were quantified by western blot with V5 antibody (lower blot). Actin serves as the loading control.

https://doi.org/10.7554/eLife.21536.020
Author response image 2
293T cells were transfected with empty vector or vector carrying the given FGFR variant, and treated with indicated AZD1480 for 24 hours.

FGFR activation was determined by detection of FGFR auto-phosphorylation (p) at Tyr653/654. Actin serves as the loading control. Control, untransfected cells. Note the efficient inhibition of FGFR activation with AZD1480, even in the case of highly active FGFR2 and FGFR3 mutants N550K and K650M, respectively.

https://doi.org/10.7554/eLife.21536.021
Author response image 3

(A) 293T cells and (B) PC12 cells were transfected with pKrox24 reporters alone (293T) or together (PC12) with vector carrying Renilla luciferase under constant promoter (pRL-TK). Cells were treated with EGF (50 ng/ml) or NGF (100 ng/ml) for 24 hours before the level of Krox24 transactivation was determined by luciferase (A) or dual-luciferase (B) assay. Values are averages from four biological replicates (each measured twice) with indicated S.D. Statistically significant differences are highlighted (Student's t-test; ***p<0.001). Results are representative for three independent experiments. The levels of EGR1 induction by EGF or NGF treatment were determined by western blot. Actin serves as the loading control.

https://doi.org/10.7554/eLife.21536.022

Additional files

Supplementary file 1

Supplementary tables containing

(A) Commercial providers of RTK activity profiling; (B) Nucleotide sequences cloned into the promoterless pGL4.17 vector expressing firefly luciferase; (C) Expression vectors used in the study; (D) Antibodies used in the study; (E) Literature survey of anti-RTK activity of BCR-ABL TKIs; (F) Primers used for reporter construction.

https://doi.org/10.7554/eLife.21536.017
Supplementary file 2

Supplementary file contains numerical data for Figure 1A,B,C,D and E; Figure 1—figure supplements 3, 4, 5A, B, 7A and B; and Figure 5—figure supplement 1.

https://doi.org/10.7554/eLife.21536.018
Supplementary file 3

Supplementary file contains numerical data for Figure 5B.

https://doi.org/10.7554/eLife.21536.019

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