Research ArticleCell Biology

The inositol phosphatase SHIP2 enables sustained ERK activation downstream of FGF receptors by recruiting Src kinases

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Science Signaling  18 Sep 2018:
Vol. 11, Issue 548, eaap8608
DOI: 10.1126/scisignal.aap8608
  • Fig. 1 FGFRs interact with SHIP2 and phosphorylate SHIP2 in cells.

    (A) 293T cells were transfected with V5-tagged wild-type (WT) FGFR3 or FGFR3-K650M together with FLAG-tagged SHIP2 and subjected to V5 or FLAG IP. Immunoprecipitates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and subjected to Western blotting (WB) with antibodies to detect FGFR3, FGFR3 phosphorylated on Tyr653 or Tyr654 (pFGFR3Y653/4), SHIP2, SHIP2-FLAG, and SHIP2 phosphorylated on Tyr986 or Tyr987 (pSHIP2Y986/7). Actin is a loading control. Note the SHIP2 phosphorylation in cells transfected with the activated FGFR3 mutant FGFR3-K650M. Immunoblotting for SHIP2 detects both endogenous and transgenically expressed SHIP2. (B) Cell-free kinase assays were carried out with purified recombinant FGFR3 and recSHIP2 proteins in the presence of adenosine 5′-triphosphate (ATP), and the reactions were separated by SDS-PAGE and subjected to Western blotting. The sample without ATP is a negative control. Phosphorylation of SHIP2 (pSHIP2) was detected with 4G10, an antibody that recognizes all phosphotyrosine residues. (C) Cell-free kinase assays were repeated in the presence and absence of the FGFR inhibitor (SU5402) and immunoblotted to show phosphorylated and nonphosphorylated forms of FGFR3 and SHIP2. Data in (A) to (C) are representative of three independent experiments. (D) Cell-free colorimetric SHIP2 phosphatase activity assay measuring SHIP2-mediated hydrolysis of phosphoinositide PtdIns(3,4,5)P3 (PIP3) to PtdIns(3,4)P2 (PIP2). Purified, recSHIP2, and recombinant FGFR3 were added to reaction buffer in the presence of absence of PIP3 as indicated. Samples without ATP are negative controls for FGFR3 activity. Significance was assessed using Welch’s t test. Each column represents three independent experiments with SD indicated. For each experiment, the values were calculated as averages from two technical duplicates, each measured three times. n.s., not significant.

  • Fig. 2 SHIP2 interacts with FGFR3 through its SH2 and SAM domains.

    (A) Schematic representation of truncated SHIP2 constructs. The V5 epitope was added to the C terminus of each construct. (B to E) The indicated SHIP2 variants were expressed with WT FGFR3 in 293T cells, immunoprecipitated (IP) using the Flag epitope tag, and analyzed by Western blotting (WB) for FGFR3 and SHIP2. Actin is a loading control. Input, total cell lysates used for IP. Data are representative of three independent experiments. IgH, immunoglobulin heavy chain. (F) Wild-type SHIP2 (SHIP2-WT)was coexpressed in 293T cells with WT FGFR3 (FGFR3-WT) or the catalytically inactive mutant FGFR3-K508M as indicated. Data are representative of three independent experiments.

  • Fig. 3 SHIP2 interacts with FGFR1 in cells.

    (A) 293T cells were cotransfected with FLAG-tagged SHIP2 and V5-tagged WT FGFR1, FGFR2, or FGFR4, and lysates were subjected to FLAG IP and Western blotting (WB) for the FGFRs and SHIP2. Data are representative of three independent experiments. (B to D) U2OS cells stably expressing FGFR1-GFP were subjected to PLA using antibodies detecting SHIP2 and GFP (B and C) or SHIP2 and FGFR1 (B and D). U2OS cells stably expressing FGFR1-BirA-HA or FGFR4-GFP were used as negative controls in (B) and (C) and (B) and (D), respectively. n = 3 independent experiments. Data are means ± SEM. Statistical significance was determined by Welch’s t test with Bonferroni’s correction of P values (n.s., P > 0.05; ***P < 0.001). Scale bar, 20 μm. Numbers in columns (B) indicate the total number of cells scored across the three independent experiments.

  • Fig. 4 FGF signaling targets SHIP2 to focal adhesions.

    (A) RCS cells were treated with FGF2 for the indicated times, and lysates were subjected to immunoblotting for Ship2 phosphorylated on Tyr986 or Tyr987 (pShip2Y986/7) and for total Ship2. Data are representative of three independent experiments. (B) Immunofluorescence images showing phosphorylated Ship2 (pShip2Y986/7) in RCS cells treated with FGF2 or not (control). Arrows indicate peripheral focal adhesions. DAPI, 4′,6-diamidino-2-phenylindole; DIC, differential interference contrast. (C) Immunofluorescence images showing pShip2Y986/7 and the focal adhesion marker vinculin (vinc.). Arrows indicate focal adhesions in RCS cells treated with FGF2. (D) Immunofluorescence images showing p130Cas phosphorylated at Tyr410 (pp130Cas) and vinculin in RCS cells treated with FGF2. Arrows indicate focal adhesions. Scale bars, 25 μm. (E) Higher magnification of boxed areas indicated in (C) and (D). Images are representative of three independent experiments. (F) Immunoblot showing Ship2 in the indicated Ship2Crispr RCS cell lines compared to WT RCS cells. Data are representative of three independent experiments. (G) Quantification of fetal bovine serum (FBS)– and FGF2-stimulated migration in WT RCS cells and the indicated Ship2Crispr cell lines. Data are means ± SEM. Statistical significance was determined by Welch’s t test with Bonferroni’s correction of P values, ***P < 0.0001. Data are representative of three independent experiments.

  • Fig. 5 Loss of SHIP2 rescues some FGF-induced cell phenotypes.

    (A) Quantification of cell proliferation, as determined by crystal violet staining, in WT RCS cells and the four indicated Ship2Crispr RCS cell lines in the presence of increasing amounts of FGF2. (B) Quantification of cell proliferation in three randomly selected RCS clones in which the Ship2 was not disrupted by CRISPR/Cas9 (2E11, 1E11, and 2B10) and the three indicated Ship2Crispr RCS cell lines. Data in (A) and (B) represent averages from eight wells ± SD. Results are representative of three independent experiments. Statistically significant differences are highlighted. ***P < 0.001, Welch’s t test. (C) Immunoblot showing total Ship2 in the indicated RCS cell lines. Results are representative of three independent experiments. (D) Immunoblot showing the cartilaginous ECM marker collagen 2 and the senescence marker caveolin in two WT and two Ship2 knockout RCS cell lines in the presence and absence of FGF2. Blot is representative of three independent experiments. (E) Alcian blue staining showing sulfated proteoglycans characteristic of cartilaginous ECM in the indicated WT and Ship2 knockout RCS cell lines in the absence and presence of FGF2. Scale bar, 200 μm. (F) Evidence for spreading in cells treated with FGF2 for 72 hours, due to the formation of actin stress fibers, visualized by phalloidin staining. Scale bar, 200 μm. (G) Immunofluorescence showing phalloidin in WT and Ship2Crispr cells in the absence and presence of FGF2. Scale bars, 20 μm. Data are representative for three independent experiments.

  • Fig. 6 Loss of Ship2 impairs Fgfr-Erk signaling.

    (A) Representative Western blot showing total and phosphorylated (p) forms of Frs2, Gab1, Akt, and Erk in WT and Ship2Crispr (Ship2a+/−) RCS cells treated with FGF2 for the indicated times. Actin is a loading control. Densitometry quantification and number of independent replicates are provided in fig. S4A. Additional analyses carried out in other Ship2Crispr cell lines are shown in figs. S3 (A to C) and S4 (C and D). (B) Quantification of Ship2 protein in three WT RCS cultures by Western blotting of lysates and serial dilutions of purified recombinant (rec) SHIP2, followed by densitometry to quantify the band intensities. The number of Ship2 molecules per cell was estimated on the basis of comparing the band intensities and number of cells in each lysate (lanes 1 to 3) with the band intensities of the purified protein samples (lanes 4 to 10). The numbers above lanes 1 to 3 indicate the number of cells represented by the lysates, and the numbers above lanes 4 to 10 represent the number of molecules of recSHIP2 per sample. On the basis of these calculations, we estimated there to be 287,546 ± 37,305 (means ± SEM) Ship2 molecules per cell. n = 9 independent RCS cultures. I.O.D., integrated optical density. (C) Ship2Crispr cells were microinjected with amounts of recombinant SHIP2 (recSHIP2) equal to about 1/10 (~25,000 molecules per cell) of the number of endogenous Ship2 molecules per cell along with a dTomato transcriptional reporter of ERK activity [pKrox24(MapErk)dTomato]. TexasRed conjugated to dextran was used as a marker for injection. The cells were then treated with FGF2 and assayed for dTomato expression. Ph2, phase contrast. Scale bar, 200 μm. (D) Quantification of dTomato expression in the two indicated Ship2Crispr cell lines treated as in (C). n = 3 independent experiments. ***P ˂ 0.001, **P ˂ 0.01, Student’s t test. (E) WT and Ship2a+/− RCS cells were treated with FGF2 for the indicated times, Frs2 was immunoprecipitated (IP) from the lysates, and the immunoprecipitates were subjected to Western blotting for Ptpn11. Blot is representative of three independent experiments.

  • Fig. 7 SHIP2 promotes the association of SFKs with FGFR3.

    (A) 293T cells were transfected with vectors encoding FLAG-tagged WT FGFR3 or the K650E activating mutant (FGFR3-K650E) together with the SFK LCK. FGFR3 was immunoprecipitated (IP) from lysates using the FLAG tag, and the immunoprecipitates were analyzed for FGFR3 and LCK by Western blotting (WB). (B and C) Bimolecular complementation assay. (B) RCS cells were transfected with vectors encoding FGFR3-Venus2 (V2) and Lyn-Venus1 (V1), fixed, and counterstained by GFP antibody to visualize expression of either part of Venus protein. ICC, immunocytochemistry; fluor., fluorescence. (C) The percentage of transfected cells showing Venus activity was calculated and plotted. n = 3 independent experiments. Data represent averages ± SEM. Scale bar, 10 μm. (D and E) RCS cells (D) and 293T cells (E) were treated with FGF2 alone or in the presence of the SRC inhibitors (inh.) AZM475271 or A419259 and analyzed by Western blotting for total and phosphorylated (p) FRS2 and ERK. Data are representative of three independent experiments. (F) Western blot showing activation (phosphorylation) of WT FGFR3 overexpressed in RCS cells in the absence and presence of the inhibitor of FGFR3 catalytic activity AZD4547 (AZD). (G and H) Immunofluorescence images showing PLAs for FGFR3 and the SFK YES tagged with YFP (YES-YFP) in RCS cells. Scale bars, 10 μm. (I) Combined results of three independent PLA experiments assessing the colocalization of FGFR3 and YES in two Ship2Crispr cell lines (Ship2c−/− and Ship2a+/−) compared to WT RCS cells. Transfections containing empty vector and a vector expressing YFP only are negative controls for the PLA assay. Data represent averages ± SEM. *P < 0.05, ***P < 0.001, Welch’s t test with Bonferroni’s correction of P values. (J) Immunofluorescence images showing PLAs testing the association of plasmid-encoded FGFR1-GFP with the endogenous SFK LYN. (K) Western blot for SHIP2 in U2OS clones with the indicated SHIP2 genotypes. (L) Combined results of three independent PLA experiments assessing the colocalization of FGFR1 and LYN in two SHIP2Crispr U2OS cell lines (SHIP2g−/− and SHIP2b−/−) compared to SHIP2+/+ U2OS cells. U2OS cells expressing FGFR4-GFP were used as a negative control for the PLA assay. Data represent averages ± SEM (**P < 0.01, ***P < 0.001, Welch’s t test).

  • Fig. 8 The inositol phosphatase activity of SHIP2 is not necessary for its association with FGFR signaling complexes or FGF-mediated ERK activation.

    (A to C) Representative Western blots showing LCK (A), LYN (B), and FRS2 (C) in SHIP2-V5 coimmunoprecipitates (IP) from 293T cells expressing the indicated combinations of V5-tagged WT SHIP2, the catalytically inactive SHIP2 triple mutant SHIP2-PD (P686A, D690A, and R691A), a SHIP2 mutant lacking the entire inositol phosphatase domain (SHIP2-ΔPS), LCK, LYN, and FRS2. Nontransfected cells or those transfected with vectors encoding only GFP serve as negative IP controls. Actin is a loading control. (D) RCS cells were treated with inhibitor of SHIP2 phosphatase activity AS1949490 (AS19) and purified FGF2 for the indicated times, and the lysates were analyzed by Western blotting for phosphorylated (p) forms of the indicated proteins. Actin and total protein are loading controls. Immuoblotting data are representative of three independent experiments. (E) Dual-luciferase assay. RCS cells were transfected with WT SHIP2 or SHIP2-ΔPS together with a firefly luciferase pKrox24 ERK reporter plasmid that is transactivated by FGF-ERK signaling and a Renilla luciferase pTK-RL control plasmid. Cells were treated with FGF2 for 24 hours before the dual-luciferase assay. Data are compiled from three independent experiments, with four biological and two technical replicates for each treatment. Bars are averages ± SD. **P < 0.01, ***P < 0.001, Welch’s t test with Bonferroni’s correction of P values. (F) Model illustrating the putative role of SHIP2 in canonical FGFR-ERK signaling. Upon being activated by binding to FGFs, FGFRs are activated and phosphorylate the adaptor protein FRS2. Although GAB1 phosphorylation can be also detected in FGF-treated cells, FRS2 is the major adaptor involved in signal relay from FGFR to the ERK pathway (17, 45, 81). Activated FRS2 and GAB1 recruit PTPN11 and SOS1 to the membrane. This activates RAS, which in turn activates ERK. Activated FGFRs also phosphorylate SHIP2 and recruit SHIP2 to the FGFR signaling complex at the cell membrane. SHIP2 recruits SFKs to the complex, which enhances FGFR-mediated phosphorylation of the adaptor proteins, activation of RAS, and ERK signaling. SHIP2 knockout effectively converts FGF-induced sustained ERK activation into the FGF-induced transient activation and rescues the cell phenotypes induced by sustained FGFR-ERK signaling (premature senescence, ECM degradation, and growth arrest). This is due to hypophosphorylation of FRS2 in SHIP2 knockout cells, resulting in diminished recruitment of PTPN11-SOS1 complexes that activate the RAS-ERK signaling module.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/548/eaap8608/DC1

    Fig. S1. Ship2 deletion or knockdown by CRISPR/Cas9 impairs Fgfr3-induced ECM degradation but not cell spreading in RCS cells.

    Fig. S2. Ship2 deletion or knockdown by CRISPR/Cas9 does not affect Fgfr2 and Fgfr3 abundance in RCS cells.

    Fig. S3. Deletion or knockdown of Ship2 impairs FGF2-mediated adaptor phosphorylation and activation of Erk.

    Fig. S4. Densitometric quantifications of Western blot analyses.

    Fig. S5. No substantial impairment of NGF- or EGF-induced changes in ERK activity in Ship2Crispr cells.

    Fig. S6. FGFR3 interacts with the SFKs FYN, LYN, FGR, and BLK.

    Fig. S7. FGR associates with both wild-type and catalytically inactive SHIP2.

    Fig. S8. Densitometric quantifications of Western blot analyses.

    Table S1. Expression vectors used in this study.

    Table S2. Antibodies used in this study.

  • This PDF file includes:

    • Fig. S1. Ship2 deletion or knockdown by CRISPR/Cas9 impairs Fgfr3-induced ECM degradation but not cell spreading in RCS cells.
    • Fig. S2. Ship2 deletion or knockdown by CRISPR/Cas9 does not affect Fgfr2 and Fgfr3 abundance in RCS cells.
    • Fig. S3. Deletion or knockdown of Ship2 impairs FGF2-mediated adaptor phosphorylation and activation of Erk.
    • Fig. S4. Densitometric quantifications of Western blot analyses.
    • Fig. S5. No substantial impairment of NGF- or EGF-induced changes in ERK activity in Ship2Crispr cells.
    • Fig. S6. FGFR3 interacts with the SFKs FYN, LYN, FGR, and BLK.
    • Fig. S7. FGR associates with both wild-type and catalytically inactive SHIP2.
    • Fig. S8. Densitometric quantifications of Western blot analyses.
    • Table S1. Expression vectors used in this study.
    • Table S2. Antibodies used in this study.

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