Different tyrosine autophosphorylation requirements in fibroblast growth factor receptor-1 mediate urokinase-type plasminogen activator induction and mitogenesis

Abstract

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1-4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1-5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1-3F) and FGFR1-Y463/583/585/766F mutant (FGFR1-4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1-3F, and FGFR1-4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

Figure 1

FGF2-mediated uPA upregulation in FGFR1-transfected L6 cells requires ERK₂ activation. (A) Parental (▪) and wt-FGFR1-transfected cells (•) were stimulated with different amounts of FGF2. Cell-associated uPA activity was evaluated after 24 h. For both cell lines, uPA activity measured in nonstimulated cells (ranging from 0.1 to 0.2 OD at 405 nm) was subtracted from all the values. (B) Northern blot analysis was performed using a murine uPA probe on total RNA extracted from parallel cultures incubated overnight with 10 ng/ml FGF2. Uniform loading of samples was judged by methylene blue staining of the filter. (C) wt-FGFR1-transfected cells were preincubated for 30 min at 37°C with no addition (1, 2) or with 100 μM PD 098059 (3) or 10 μM SB 210313 (4) before addition of 100 ng/ml FGF2 (2–4). After 24 h, cell-associated uPA activity was measured. uPA activity in control nontransfected L6 cells incubated under the same experimental conditions was subtracted from all the values. (D) Parallel cultures were lysed 20 min after FGF2 addition, and cell lysates were probed with anti-ERK₂ antibody in a Western blot. *Erk-2 indicates the phosphorylated protein.

Figure 2

uPA-inducing capacity of different FGFR1 mutants. L6 myoblasts were independently transfected with wt-FGFR1 or with the FGFR1 mutants described in the bottom panel. In each column, an asterisk or asterisks mark the tyrosine autophosphorylation site(s) mutagenized to phenylalanine in the different mutants. Each cell line was stimulated with 100 ng/ml FGF2, and 24 h later cell-associated uPA activity was evaluated (top panel). For each cell line, uPA activity measured in the corresponding nonstimulated cells (ranging from 0.1 to 0.2 OD at 405 nm) was subtracted. Results represent the mean ± SD of two independent experiments in triplicate. For each mutant, similar results were obtained for two independent clones.

Figure 3

Dissociation of uPA upregulation from mitogenicity in L6 transfectants. (A) wt-FGFR1 (•), FGFR1–3F (▴), and FGFR1–4F (▾) cell lines were stimulated with FGF2. Cell-associated uPA activity was evaluated after 24 h. For each cell line, uPA activity measured in corresponding nonstimulated cells (ranging from 0.1 to 0.2 OD at 405 nm) was subtracted from all the values. (B) Parallel cultures were incubated overnight in the absence or presence of 10 ng/ml FGF2. Then, total RNA was extracted and analyzed by Northern blot using a murine uPA probe. Uniform loading of the gel was judged by methylene blue staining of the filter (28S). (C) Parental L6 myoblasts (1), wt-FGFR1 (2), FGFR1–3F (3), and FGFR1–4F (4) cell lines were seeded in 48-well plates, starved for 48 h in the presence of 0.1% FCS, and treated with 30 ng/ml FGF2 (gray bar) or vehicle (open bar) for 20 h. Then [³H]thymidine was added, and 6 h later samples were collected and [³H]thymidine incorporation was evaluated. Data are the mean ± SD of three determinations.

Figure 4

Signal transduction analysis of FGFR1 mutants. Parental L6 myoblasts, wt-FGFR1, FGFR1–3F, and FGFR1–4F cells were nonstimulated (−) or treated (+) either for 5 min (A, B, and C) or 20 min (D, E, and F) with 100 ng/ml FGF2. (A) Anti-FGFR1 immunoprecipitates were analyzed for FGFR1 expression using anti-FGFR1 antibodies. (B) Anti-FGFR1 immunoprecipitates were analyzed for tyrosine phosphorylation using antiphosphotyrosine (P-Tyr) antibodies. (C) Anti-Shc immunoprecipitates were analyzed for tyrosine phosphorylation. (D) Cell lysates (50 μg of total protein) were analyzed with anti-ERK₂ antibodies. *Erk-2 indicates the phosphorylated protein. (E) Anti-ERK₂ immunoprecipitates were analyzed for kinase activity using myelin basic protein (MBP) as substrate. (F) Cell lysates (50 μg of total protein) were analyzed with anti-JunD antibodies. *JunD indicates the phosphorylated protein. N.D., not determined.

Figure 5

Residues Y463 and Y730 are implicated in uPA upregulation but not mitogenesis by FGF2. (A) wt-FGFR1 (1), FGFR1-Y583/585/730F (2), and FGFR1-Y463/730F (3) transfectants were assayed for cell-associated uPA activity after a 24-h incubation in the absence (open bar) or presence (gray bar) of 100 ng/ml FGF2. (B) wt-FGFR1 and FGFR1-Y463/730F transfectants were seeded in 48-well plates, starved for 48 h in the presence of 0.1% FCS, and then treated with no addition (open bar), 100 ng/ml FGF2 (gray bar), or 10% FCS (dashed bar) for an additional 20 h. Then, [³H]thymidine was added, and 6 h later samples were collected and [³H]thymidine incorporation was evaluated. (C) Parallel cultures were lysed 20 min after FGF2 addition, and cell lysates (30 μg of protein) were probed with antiphosphorylated ERK_1/2 antibody in a Western blot. (D) Parental L6 cells (○), wt-FGFR1 (•), and FGFR1-Y463/730F (▴) transfectants were seeded in 96-well plates at 20,000 cells/cm² in DMEM plus 10% FCS. After 24 h of serum starvation in DMEM plus 0.1% FCS, cells were stimulated with increasing concentrations of FGF2. Cells were counted after 2 d. For each mutant, similar results were obtained for two independent clones in all the assays.