*2.2. In Vitro Phosphorylation of FRS2 by p38*α *Kinase*

Since the addition of either p38 inhibitor or p38 activator resulted in FRS2 mobility changes in SDS-PAGE and Phos-Tag PAGE, we hypothesized that p38, in addition to Erks, is also able to phosphorylate FRS2. To verify such possibility, we performed in vitro phosphorylation using recombinant FRS2 and recombinant active form of p38α kinase. Using autoradiography we observed a clear band corresponding to phosphorylated FRS2 when active p38α was present during the reaction (Figure 1d, lanes 3 and 4, two parallel samples). FRS2 phosphorylation was dependent on p38 activity, since in the presence of the specific p38 inhibitor, SB203580, there was no trace of radioactivity (Figure 1d, lane 5). Interestingly, we did not observe any differences in the efficiency of phosphorylation (assessed by intensity of bands) of FRS2 in the case of p38α and both Erks (Erk1/2) (Figure 1d). These data indicate that p38 can phosphorylate FRS2.

#### *2.3. Synergistic E*ff*ect of MEK1*/*2 and p38 Inhibitors on Kinetics of FGF1-Induced Signaling*

Next, we studied the impact of p38 activity on the kinetics of signaling cascades activated by FGF1 in NIH3T3 (Figure 2a). Experiments were performed in the presence of brefeldin A (2 μg/mL) to prevent the appearance of newly synthesized receptors [7]. In the presence of 5 μM of SB203580, p38 inhibitor (Figure 2a, lanes 9–13), or 10 μM anisomycin, p38 activator (Figure 2a, lanes 16–20), we did not observe significant differences in the intensity of FRS2 phosphorylation and FRS2 mobility, as compared to untreated cells. However, when the cells were simultaneously treated with 20 μM of MEK1/2 inhibitor (U0126) and either p38 inhibitor (SB203580) or p38 activator (anisomycin), the phosphorylation pattern of FRS2 varied, as well as the duration of FGFR1 tyrosine phosphorylation. We observed a synergistic effect of SB203580 and U0126. When activity of both kinds of MAP kinases (Erks and p38) were blocked, not only was the intensity of the band corresponding to tyrosine-phoshorylated FGFR1 stronger and lasted longer (Figure 2a, lanes 30–34), but also the electrophoretic mobility shift of FRS2 was prolonged, as compared to the inhibition of MEK1/2 with U0126 alone (Figure 2a, lanes 23–27). Fifteen minutes after FGF1 stimulation, FRS2 migrated faster in the presence of U0126, as well as in the presence of U0126 and SB203580, than in the absence of inhibitors. In the course of the experiment, the FRS2 band shifted upwards. When Erks and p38 were blocked at the same time (Figure 2a, lanes 30–34), we observed a more gradual change in the position of the FRS2 band than in the presence of U0126 alone (Figure 2a, lanes 23–27). In contrast to the effect of p38 inhibition, hyper-activation of p38 with 10 μM anisomycin used in the combination with 20 μM U0126 compensated, to some extent, the lack of Erk activity (Figure 2a, lanes 37–41). In the presence of anisomycin and U0126, the FRS2 band was less down-shifted after 15-min treatment with FGF1 and moved to the direction of higher molecular masses much faster over time than in the case of cells treated only with U0126. We confirmed all these findings in U2OS-R1 cells (Figure 2b). These data demonstrate that Erks and p38 regulate phosphorylation status of FRS2, which in turn modulates kinetics of FGFR1 signaling (Figure 2b).

**Figure 2.** The crosstalk between p38 and Erk1/2 in downregulation of FGF1-induced signaling. Serum-starved (**a**) NIH3T3 and (**b**) U2OS-R1 cells were pretreated for 30 min with or without 20 μM U0126, 5 μM SB203580 and 10 μM anisomycin, and then stimulated with the growth factor in the presence of heparin (10 U/mL) and brefeldin A (2 μg/mL) for different time points. Cells were lysed, and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 as a loading control. Anti-phospho-Erk1/2 (p-Erk1/2) and anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38) were used to control the effect of U0126 (MEK inhibitor) and anisomycin (p38 activator). Representative experiments are shown, n = 3. The graphs present quantification of bands from panel b corresponding to phospho-FGFR1 (Tyr653/Tyr654) and phospho-FRS2 (Tyr196) normalized to loading control (Hsp90) and expressed as a fraction of the maximal response in the absence of inhibitor. Data are means ± SD of three independent experiments; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.
