Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2

Abstract

Fibroblast growth factor 2 (FGF2) is a potent mitogen promoting both tumor cell survival and tumor-induced angiogenesis. It is secreted by an unconventional secretory mechanism that is based upon direct translocation across the plasma membrane. Key steps of this process are (i) phosphoinositide-dependent membrane recruitment, (ii) FGF2 oligomerization and membrane pore formation, and (iii) extracellular trapping mediated by membrane-proximal heparan sulfate proteoglycans. Efficient secretion of FGF2 is supported by Tec kinase that stimulates membrane pore formation based upon tyrosine phosphorylation of FGF2. Here, we report the biochemical characterization of the direct interaction between FGF2 and Tec kinase as well as the identification of small molecules that inhibit (i) the interaction of FGF2 with Tec, (ii) tyrosine phosphorylation of FGF2 mediated by Tec in vitro and in a cellular context, and (iii) unconventional secretion of FGF2 from cells. We further demonstrate the specificity of these inhibitors for FGF2 because tyrosine phosphorylation of a different substrate of Tec is unaffected in their presence. Building on previous evidence using RNA interference, the identified compounds corroborate the role of Tec kinase in unconventional secretion of FGF2. In addition, they are valuable lead compounds with great potential for drug development aiming at the inhibition of FGF2-dependent tumor growth and metastasis.

Keywords: FGF2; Tec kinase; fibroblast growth factor (FGF); phosphoinositide; protein phosphorylation; protein secretion; protein translocation; protein translocation across membranes; unconventional protein secretion.

FIGURE 1.

The catalytic SH1 domain of Tec kinase directly interacts with FGF2 as analyzed by biochemical pull-down experiments. A, schematic depiction of the domain structure of Tec constructs used in this study. B, pull-down experiments using recombinant FGF2 covalently coupled to epoxy beads as analyzed by SDS-PAGE and Coomassie protein staining. FGF2-conjugated epoxy beads were incubated with each of the four Tec kinase-derived constructs, GST-PH-TH (47.2 kDa; lanes 2 and 3), GST-SH3-SH2 (47.9 kDa; lanes 4 and 5), SH1 (32.9 kDa; lanes 6 and 7), and GST-NΔ173 Tec (80.2 kDa; lanes 8 and 9). Because all constructs were used as GST fusion proteins (with the exception of the SH1 kinase domain), binding of GST alone (26.5 kDa; lanes 10 and 11) to FGF2-conjugated epoxy beads was taken as a negative control. For each construct, bound (100%) and unbound (1%) fractions were loaded. Lane 1, molecular mass markers (M; PageRuler prestained protein ladder: 140, 115, 80, 65, 50, 40, 30, 25, 15, and 10 kDa). The gel shown is representative of five independent experiments. C, quantification of FGF2 binding to the various Tec constructs depicted in A. Coomassie-stained SDS gels were analyzed using the LI-COR Biosciences Odyssey infrared imaging system. The intensity of each band was quantified using Image Studio software (version 2.1.10). For each construct, binding efficiency was calculated relative to unbound material. A comparison of all constructs was conducted, defining FGF2 binding efficiency toward GST-NΔ173 Tec as 100%. The statistical analysis was based upon five independent experiments. Error bars, S.D.

FIGURE 2.

Determination of the dissociation constant of the interaction between FGF2 and various forms of Tec kinase based upon fluorescence polarization. A, fluorescence polarization experiments were conducted using fluorescein-labeled FGF2. FGF2 (at a constant concentration of 50 nm) was incubated with increasing concentrations (0–20 μm) of the various Tec constructs indicated. Following incubation for 3 h at room temperature, changes in polarization (ΔPolarisation) (in millipolarization units (mP)) were recorded. A non-linear regression analysis was conducted (GST-NΔ173 Tec (black circles), n = 8; SH1 kinase domain (green squares), n = 5; GST-PH-TH (blue triangles), n = 3; GST-SH3-SH2 (red triangles), n = 3; GST (orange rhombuses), n = 5), and S.E. values were calculated. As detailed under “Experimental Procedures,” assuming a binding stoichiometry of 1:1, dissociation constants were calculated to be 1.434 ± 0.55 μm (S.E.) for GST-NΔ173 Tec and 1.032 ± 0.29 μm (S.E.) for the SH1 kinase domain of Tec. B, competition experiments for the interaction between SH1 kinase domain of Tec GST-NΔ173 Tec and fluorescein-labeled FGF2. Experiments were conducted as described above. Conditions in the absence and presence of unlabeled FGF2 (50 μm) were compared. All data points were normalized based on measurements on fluorescein-labeled FGF2 alone. Errors are given as S.E. An unpaired and one-tailed t test was conducted to assess statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). C, competition experiments for the interaction between GST-NΔ173 Tec and fluorescein-labeled FGF2. Experiments were conducted as described above. Conditions in the absence and presence of unlabeled FGF2 (50 μm) were compared. All data points were normalized based on measurements on fluorescein-labeled FGF2 alone. Error bars, S.E. An unpaired and one-tailed t test was conducted to assess statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

FIGURE 3.

A protein-protein interaction assay designed to screen small molecule libraries for compounds inhibiting FGF2 binding to Tec kinase. The direct interaction between FGF2 and Tec kinase was quantified using Alpha® technology (red spheres). A His-tagged form of FGF2 (62.5 nm) and GST-NΔ173Tec (31.25 nm) were used along with glutathione-coated donor and Ni-NTA-coated acceptor beads as explained under “Experimental Procedures.” As a specificity control, an unrelated protein pair, His-tagged MBP-CARP and GST-Titin (black spheres), was used. Alpha® signals were measured in the presence of increasing concentrations of untagged NΔ25FGF2, a competitor for binding of His-tagged FGF2 to GST-NΔ173 Tec. Alpha® signals are expressed as percentage of the median of the maximal Alpha® signal (tagged proteins in the absence of the NΔ25FGF2 competitor), which was set to 100% in each independent experiment. Data points represent the mean of eight independent experiments, each of which consisted of three technical replicates. Experimental deviations are expressed as S.E. (error bars). Data were fitted with a non-linear regression model. The K_D of the GST-NΔ173 Tec-N25ΔFGF2 complex was calculated to be 0.63 ± 0.033 μm (S.E.) (r² = 0.9986).

FIGURE 4.

Chemical structures of active (compounds 6, 14, and 21) and inactive (compounds 18 and 19) compounds. EMBL IDs are provided as a reference to the internal database of the Chemical Biology Core Facility at EMBL Heidelberg (58).

FIGURE 5.

Determination of dose-response curves for active (compounds 6, 14, and 21) versus inactive (compounds 18 and 19) compounds. Alpha® protein-protein interaction assays with His-tagged FGF2 (125 nm) and GST-NΔ173 Tec (30 nm) (blue spheres) as well as His-tagged MBP-CARP (20 nm) and GST-titin (20 nm) (red squares) were conducted as described in the legend to Fig. 3 and under “Experimental Procedures.” Additionally, as a technical control, a single fusion protein containing both a GST and a His tag was used (20 nm; black triangles). Dose-response curves were recorded with the three active compounds (compounds 6, 14, and 21) as well as the two control compounds (compounds 18 and 19) shown in Fig. 4 (58). For each compound, four independent experiments (each of which was conducted in three technical replicates) were performed. Data points were fitted using the non-linear regression function log (inhibitor) versus response − variable slope (four parameters). Data were evaluated using GraphPad Prism (version 5 for Macintosh OS X). Error bars, S.D. A, dose-response curves of compound 6. IC₅₀ (His-FGF2/GST-NΔ173 Tec) = 8.9 ± 1.1 μm (S.E.). B, dose-response curves of compound 14. IC₅₀ (His-FGF2/GST-NΔ173 Tec) = 7.0 ± 1.1 μm (S.E.). C, dose-response curves of compound 21. IC₅₀ (His-FGF2/GST-NΔ173 Tec) = 11.7 ± 1.0 μm (S.E.). D, dose-response curves of compound 18. E, dose-response curves of compound 19.

FIGURE 6.

Small molecule inhibition of Tec kinase-mediated tyrosine phosphorylation of FGF2. FGF2 tyrosine phosphorylation and Tec kinase autophosphorylation were reconstituted with purified components based on Western analysis using anti-phosphotyrosine antibodies directed against an FGF2-derived phosphopeptide. In vitro phosphorylation experiments were conducted in the absence (1% DMSO mock control) and presence of the small molecule inhibitors (compounds 6, 14, and 21; 50 μm in 1% DMSO) and control compounds (compounds 18 and 19; 50 μm in 1% DMSO) introduced in Figs. 4 (58) and 5. Fluorescent secondary antibodies were used to detect antigens, employing the LI-COR Odyssey imaging platform. M, PageRuler prestained protein ladder (140, 115, 80, 65, 50, 40, 30, 25, 15, and 10 kDa). For details, see “Experimental Procedures.” A, representative Western analysis using antibodies recognizing both phosphorylated FGF2 (FGF2(P)) and Tec kinase in its autophosphorylated form (GST-NΔ173 Tec(P)). B, Coomassie-stained SDS gel corresponding to the Western analysis shown in A. C, quantification and statistical analysis of five independent experiments (corresponding to the example shown in A and B) using the LI-COR Odyssey imaging platform. Following background normalization, the average of FGF2 tyrosine phosphorylation under mock conditions was defined as 100% activity. Error bars, S.D. A one-tailed and unpaired t test was conducted to test significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). NS, absence of significant differences. D, representative Western analysis using His-tagged variant forms of FGF2 (WT, Y81F, Y111F, and Y123F, as indicated) to characterize the anti-phosphotyrosine-FGF2 antibody used in A. E, Coomassie-stained SDS gel corresponding to the Western analysis shown in D. F, quantification and statistical analysis of six independent experiments (corresponding to the example shown in D and E) using the LI-COR Odyssey imaging platform. Following background normalization, the average of tyrosine phosphorylation of FGF2 wild-type was defined as 100% activity. S.D. values are shown. A one-tailed and unpaired t test was conducted to test significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). NS, absence of significant differences.

FIGURE 7.

Selectivity of small molecule inhibitors toward Tec kinase-mediated tyrosine phosphorylation of FGF2. FGF2 and STAP1 tyrosine phosphorylation as well as Tec kinase autophosphorylation were analyzed as described in the legend to Fig. 6 and under “Experimental Procedures.” In vitro phosphorylation experiments were conducted in the absence (1% DMSO mock control) and presence of the small molecule inhibitors (compounds 6, 14, and 21; 50 μm in 1% DMSO) and control compounds (compounds 18 and 19; 50 μm in 1% DMSO) introduced in Figs. 4 (58) and 5. Western analysis was conducted using anti-phosphotyrosine antibodies (anti-Tyr(P)-FGF2 for FGF2 and Tec as well as anti-Tyr(P)-4G10 for STAP1). M, PageRuler prestained protein ladder: 140, 115, 80, 65, 50, 40, 30, 25, 15, and 10 kDa. Fluorescent secondary antibodies were used to detect and quantify antigens using the LI-COR Odyssey imaging platform. In D–F, following normalization based upon background signals detected in the absence of Tec kinase or ATP, the average of the signals detected under mock conditions was defined as 100% activity. Error bars, S.D. A one-tailed and unpaired t test was conducted to assess statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). ns, absence of significant differences. A, Tec kinase-mediated tyrosine phosphorylation of FGF2 in the absence and presence of active and inactive compounds (50 μm each). B, Tec autophosphorylation in the absence of a substrate measured in the absence and presence of active and inactive compounds (50 μm each). C, Tec kinase-mediated tyrosine phosphorylation of STAP1 in the absence and presence of active and inactive compounds (50 μm each). Following protein transfer to PVDF, membranes were cut to stain the upper part with anti-Tyr(P)-FGF2 antibodies (to detect Tec autophosphorylation) and the lower part with anti-Tyr(P)-4G10 antibodies (to detect phosphorylated STAP1). D, quantification of the Western analysis shown in A. E, quantification of the Western analysis shown in B. F, quantification of the Western analysis shown in C.

FIGURE 8.

Small molecule protein-protein interaction inhibitors of the Tec-FGF2 complex block tyrosine phosphorylation of FGF2 in cells. CHO cells were induced with doxycycline to express FGF2-GFP fusion proteins under the conditions indicated (see “Experimental Procedures” for details). All compounds were used at a final concentration of 50 μm in 0.5% DMSO. The mock controls correspond to 0.5% DMSO in the absence of compound. Following cell lysis, FGF2-GFP fusion proteins were affinity-purified employing GFP trap magnetic beads. Proteins were eluted with SDS sample buffer and subjected to SDS-PAGE. Following Western blotting, the LI-COR imaging platform was used to quantify both tyrosine phosphorylation (4G10) and total FGF2-GFP (anti-FGF2). In addition to samples derived from cells (lanes 2–4 and 9–12), recombinant FGF2-GFP (2 ng) and in vitro phosphorylated FGF2-GFP (2 ng) were used as negative and positive controls for FGF2 tyrosine phosphorylation (lanes 5 and 6 and lanes 13 and 14). 165 ng of recombinant FGF2-GFP (an amount comparable with the material isolated from cells) were used to quantify the background of the 4G10 anti-phosphotyrosine antibody (lanes 7 and 15). In lanes 1 and 8, marker proteins of 50, 40, and 30 kDa are shown. A, quantitative Western analysis of affinity-purified FGF2-GFP fusion proteins from cells cultivated under the conditions indicated. B, quantification and statistical evaluation of the Western analysis shown in A. FGF2 phosphotyrosine signals (4G10) were normalized by total FGF2-GFP. The statistical analysis was based upon four independent biological replicates. Error bars, S.D. A one-tailed and unpaired t test was conducted to reveal significant differences between the mock control and conditions in the presence of compounds (*, p < 0.05; **, p < 0.01). ns, absence of significant differences.

FIGURE 9.

Small molecule inhibitors blocking Tec kinase mediated tyrosine phosphorylation of FGF2 inhibit unconventional secretion of FGF2 from cells. A stable CHO cell line expressing a FGF2-GFP fusion protein in a doxycycline-dependent manner was used to quantify FGF2 transport to cell surfaces in the absence and presence of the small molecule inhibitors introduced in Figs. 4–7 (58). As detailed under “Experimental Procedures,” following cultivation of cells in the presence of compounds as indicated and induction of FGF2-GFP expression, proteins localized to the cell surface were biotinylated with a membrane-impermeable reagent. Following quenching of the biotinylation reagent, biotinylated proteins were purified using streptavidin beads. Following SDS-PAGE analyzing both the total lysate (input termed Cells; 1.7%) and the biotinylated cell surface fraction (33%), a Western analysis was conducted to detect the secreted population of FGF2-GFP. Using appropriate fluorescent secondary antibodies, both FGF2-GFP and GAPDH (used as a control protein restricted to the intracellular space) were quantified using the LI-COR Odyssey imaging platform. FGF2-GFP secretion quantified under mock conditions (0.5% DMSO) was defined as 100% secretion efficiency. Error bars, S.D. values from three independent experiments, each of which was conducted in two technical replicates. A two-tailed and unpaired t test was conducted to assess statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). ns, absence of significant differences. A, quantification of FGF2-GFP secretion from cells in the absence and presence of compound 6 (active). B, quantification of FGF2-GFP secretion from cells in the absence and presence of compound 14 (active). C, quantification of FGF2-GFP secretion from cells in the absence and presence of compound 21 (active). D, quantification of FGF2-GFP secretion from cells in the absence and presence of compound 18 (inactive). E, quantification of FGF2-GFP secretion from cells in the absence and presence of compound 19 (inactive).

FIGURE 10.

Small molecule inhibitors blocking Tec kinase-mediated tyrosine phosphorylation of FGF2 do not exert apparent pleiotropic effects on cell viability and proliferation. CHO cells expressing mCherry fused to a nuclear localization signal were used to monitor potential pleiotropic effects on cell viability and proliferation of the compounds introduced in Fig. 4 (58). Cell proliferation was monitored by absolute counting of fluorescent nuclei using an IncuCyte Zoom live cell imaging microscope (Essen Biosciences). As a starting density, 1 × 10⁴ cells were cultivated per experimental condition in 96-well plates. The actual cell number measured at t = 0 was set to 100%. Cell proliferation was monitored at 37 °C for 72 h under mock conditions (0.5% DMSO) as well as in the presence of 10, 25, and 50 μm concentrations of compounds 6 (A), 14 (B), 21 (C), 18 (D) and 19 (E). Cell numbers were determined in intervals of 2 h. The experiments shown are representative of a total of four biological replicates, each of which contained three technical replicates for every single data point. Error bars, S.D.