Abstract
FGFR2-expressing human cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In nonstimulated cells, the SH3 domain (and not the SH2 domains) of Plcγ1 directly competes for a binding site at the very C terminus of FGFR2 with the C-terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits Plcγ1 access to the receptor. Recruitment of Plcγ1 in this way is sufficient to upregulate phospholipase activity. This results in elevated phosphatidylinositol 4,5-bisphosphate turnover and intracellular calcium levels, thus leading to increased cell motility and promotion of cell-invasive behavior in the absence of extracellular receptor stimulation. Therefore, metastatic outcome can be dictated by the constitutive competition between Grb2 and Plcγ1 for the phosphorylation-independent binding site on FGFR2.
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Acknowledgements
J.E.L. is funded by the G. Harold and Leila Y. Mathers Charitable Foundation. R.G.O. is funded through US National Institutes of Health DK070950. We thank H. Xin for assistance with Figure 1. Full-length HA-Plcγ1 was a generous gift from J. Sondek (University of North Carolina School of Medicine).
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Z.T., Z.A., C.-C.L., F.A.M., L.J.S., P.G.L., P.J. and J.B. carried out experiments and analyzed data. Z.T., R.G.O., M.B. and J.E.L. conceived the experiments. Z.T. and J.E.L. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Plcγ1 binding to FGFR2 is independent of the phosphorylation state.
(a) Western blot showing the efficiency of knockdown of Grb2 (G2i cells) compared to cells transfected with scrambled shRNA (Ci cells). Cell lysates from serum starved A431 Ci and G2i cells were utilized for this experiment. The membrane was probed for Grb2 and total Plcγ/β-actin as loading controls. (b) Western blot showing the phosphorylation state of Plcγ1 in unstimulated and FGF9-stimulated cells. Cell lysates from serum starved Ci, G2i and G2i cells with 0.5 μg Grb2 knocked in (rescue study) were used in this experiment. The membrane was probed for phosphorylated Plcγ1, total Plcγ1 (loading control) and Grb2 to show the efficiency of knockdown/knock in. (c) Western blot showing the efficiency of transfection of RFP-tagged constructs (SH223, SH3, RFP empty vector) in HEK293T control and knockdown cells. (d) HEK293T Ci and G2i cells stably expressing FGFR2-GFP were transfected with SH223-RFP, serum starved overnight then treated with FGF9 for 30 minutes. Cells were washed and lysed before immunoprecipitation with anti-GFP antibody. Following western blot analysis, membranes were probed for GFP (as a control) and RFP to detect association of FGFR2 with Plcγ1 SH223 domain. (e) Cells transfected with SH223-RFP or RFP alone were used to detect binding to FGFR2-GFP by FLIM. Reduced fluorescence lifetime (left shift of peaks in right hand column) indicates complex formation.
Supplementary Figure 2 Plcγ1SH3-mediated phospholipase activity is concentration dependent and independent of phosphorylation.
(a) Cell lysates from Ci and parental HEK293T cells were used in a western blot to show the negligible level of FGFR2 expression in the parental cells (upper panel). Cell lysates from serum starved parental cells with and without over expressed Plcγ1 were used in a western blot to show the efficiency of transfection (lower panel). Blots were probed for total FGFR2, total Plcγ1 and β-actin. (b) Plcγ1 is active in the absence of phosphorylation and intramolecular SH2 domain interactions. Five point mutations where made in full length HA-Plcγ1 (Y771F, Y775F, Y783F, Y977F, Y1253F; TM; http://www.phosphosite.org). Two point mutations were made in CSH2 and NSH2 of full length HA-Plcγ1 (R586A and R694A; DM). HEK293T cells were transiently transfected with DM, TM or left untransfected (NT). Cells were serum starved and lysed before western blot analysis to detect the efficiency of transfection. The membrane was probed by the following antibodies: anti-HA, anti-total Plcγ1 and anti- β-actin (as a loading control). (c) Cellular lipase assay was performed on cell lysates from untransfected, DM transfected and TM transfected serum starved HEK293T cells. [3H]IP3 to [3H]Ptldlns(4,5)P2 ratios were normalized against control cells. Both mutants are able to turnover lipid. Elevated concentrations of Plcγ1 mutants are capable of out-competing endogenous Grb2 for binding to FGFR2. In G2i cells the difference in turnover was less dramatic presumably because the competition for binding to the receptor is reduced in the absence of Grb2. Error bars represent standard deviation. (d) HEK293T Ci and G2i cells were either left untransfected or transfected with 0.5 μg of Plcγ1. Cells were serum starved overnight then lysed before western blot analysis where the membranes were probed by anti-total Plcγ1 and anti- β-actin (as a loading control). (e) Lipase assay on untransfected and HA-Plcγ1 transfected serum starved HEK293T cells. The ratio of [3H]IP3 to [3H]Ptldlns(4,5)P2 was calculated after scintillation counting. The graph represents the average of three independent experiments following a 24 h incubation period with the substrate. Values were normalized against untransfected control cells and the standard deviation was determined. (f) HEK293T control cells were left untransfected whereas the knockdown cells were either untransfected or transfected with increasing amounts of myc-tagged Grb2 (0.5 μg and 1 μg). Western blot analysis was carried out using cell lysates from serum starved cells and probed for Grb2, total Plcγ1 and β-actin (the last two were used as loading controls).
Supplementary Figure 3 Plcγ1 DM is functionally active and is responsible for increased cellular migration.
(a) Control cells transfected with DM construct were used for scratch wound healing assay. Cells were treated with 1% serum. Images were taken at 0 h and 6 h. (b) Percentage wound healing from three independent experiments of untransfected and DM transfected control cells were measured then averaged and normalized against control cells. Error bars represent standard deviation. (c) HEK293T Ci cells transfected with DM construct were loaded with Fura 2-AM in Hanks' buffer before measurement of the basal release of calcium. Intracellular calcium concentration ([Ca2+]i) and ratio F340/380 emission ratios are plotted. Arrows indicate the time when ionomycin was added. (d) Calcium release in HEK293T Ci cells (untransfected and DM transfected) was compared and the first two minutes of measurement were averaged based on three independent experiments. Results are shown as [Ca2+]i and F340/380 emission ratio. Error bars represent standard deviation. (e) MTT assay performed on Ci (untransfected and transfected with DM construct) based on 3 independent experiments. Standard deviation is shown on the graph as error bars.
Supplementary Figure 4 Plcγ1 is responsible for invasive behavior in cells.
(a) HEK293T Ci and G2i cells were treated with 1% serum containing 9 μM U73122 and along with Plcγ1 knockdown cells (Pγi) were used in a scratch wound healing assay. Images were taken at the beginning of the experiment and after 6 hours. (b) Distance traveled by the untreated and U73122 treated cells was quantified in three independent experiments. The normalized percentage of wound closure was calculated. Error bars represent standard deviation. (c) Histograms on left: Averages of invasive untreated and U73122 treated HEK293T, LoVo and ROS cells were calculated based on triplicates. Cells in four microscopic fields were counted for every sample. Standard deviation is shown on the graph as error bars. Histograms on right: MTT assays were performed on untreated cells and cells treated with U73122 to detect if there are any changes in proliferation/apoptosis upon knocking down Grb2, Plcγ1 or transfection with scrambled shRNA and following the administration of the U73122. Averages were based on three independent measurements and error bars indicate the calculated standard deviation. Results on the Y-axis were calculated in relative fluorescent units (RFU). (d) Western blot showing the efficiency of Grb2 and/or Plcγ1 knockdown in HEK293T, LoVo, and ROS cells. Ci cells were used as controls in every case. The membranes were probed for total Plcγ1, Grb2, and β-actin. (e) Cell lysates from serum starved HEK293T Ci, G2i cells and LoVo cells were used for western blot analysis. The membranes were probed for total Plcγ1 and Grb2 to compare their level of expression in these two different cell lines. β-actin was used as loading control. (f) LoVo cells were left untransfected or transfected with myc-tagged Grb2 (0.5ug, and 1 ug). Cells were serum starved overnight then lysed. Cell lysates were used for western blot analysis. Blots were probed for Grb2 to check for the efficiency of transfection. Total Plcγ1 and tubulin were used as loading controls. (g) Left panel: Ci, G2i and Pγi HEK293T and ROS cells along with Ci and Pγi LoVo cells (LoVo cells endogenously possess a relatively low level of Grb2 expression as shown in (e)) were used in an invasion assay. Middle panel: HEK293T and ROS cells (Ci and G2i) without treatment or with U73122 treatment added in the upper and lower chambers to a final concentration of 9 μM were used in an invasion assay. Right panel: LoVo cells (untransfected with shRNA) left untreated or treated with U73122 were used in an invasion assay. In all cases cells were incubated for 24 hours, fixed, stained, mounted onto microscopic slides and pictures were taken at 20X magnification.
Supplementary Figure 5 Invasive behavior in cells is FGFR2 dependent.
(a) Parental cells with low levels of FGFR2 expression were used for the generation of stable knockdowns of Grb2 and Plcγ1 to generate PG2i and PPγi cells. Scrambled shRNA was transfected to generate PCi control cells. Serum starved cells were lysed and used in western blot analysis to detect efficiency of the knockdown by probing for Grb2 and Plcγ1. β-actin was used as a loading control. (b) PCi, PG2i and Pγi cells were used in an invasion assay carried out for 24 hrs. Scale bar 160 μm. (c) Averages of invasive PCi, PG2i and Pγi cells were calculated based on triplicates. Cells in four microscopic fields were counted for every sample. Standard deviation denoted by error bars is shown on the graph. (d) MTT assay was performed on PCi, PG2i and Pγi. Averages were based on 3 independent experiments. Standard deviation is shown on the graph as error bars.
Supplementary Figure 6 Plcγ1 localizes on migratory structures in G2i cells.
(a) HEK293T Ci and G2i cells stably expressing FGFR2-GFP were transiently transfected with HA-Plcγ1. Serum starved cells were treated with primary HA-antibody labeled with Alexa Fluor 555. Microscopy was used to detect colocalization of Plcγ1 on FGFR2 (yellow). High magnification view of co-localization on migratory structures (lower far right panel) (b) HEK293T Ci and G2i cells were detached and treated with 1% serum for 2 h then stained with phalloidin to detect actin polymerization and Hoechst (nuclear stain). Intensity of the phalloidin staining and change in G2i cell morphology corresponds to increase in migratory ability compared to Ci cells.
Supplementary Figure 7 Actin polymerization is higher in G2i compared to Ci cells.
(a), (b) HEK293T Ci and G2i cells were counted, fixed, and stained with phalloidin after they had been detached and treated with 1% serum for 2 h. Flow cytometry was used for the quantification of the extent of actin polymerization in cells. (c) Results of flow cytometry from three independent experiments were averaged and normalized against control cells. Error bars represent standard deviation. (d) Controls for phospholipase in vitro reconstitution assays. The ratio of [3H]IP3 to [3H]PIP2 was determined and averaged based on three independent experiments. Values were normalized against that of Plcγ1 with kinase dead receptor. Standard deviation was calculated and is shown on graph.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–8 and Supplementary Table 1 (PDF 5785 kb)
Supplementary Video 1
Cell motility is increased in the absence of Grb2–Ci cells. (MP4 5938 kb)
Supplementary Video 2
Cell motility is increased in the absence of Grb2–G2i cells with U73122. (MP4 5940 kb)
Supplementary Video 3
Cell motility is increased in the absence of Grb2–G2i cells. (MP4 6579 kb)
Supplementary Video 4
Cell motility is increased in the absence of Grb2–Pγi cells. (MP4 5804 kb)
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Timsah, Z., Ahmed, Z., Lin, CC. et al. Competition between Grb2 and Plcγ1 for FGFR2 regulates basal phospholipase activity and invasion. Nat Struct Mol Biol 21, 180–188 (2014). https://doi.org/10.1038/nsmb.2752
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DOI: https://doi.org/10.1038/nsmb.2752
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