*2.4. The E*ff*ect of p38 Kinase on Erk1*/*2 Activity*

Finding a synergistic effect of the inhibition of both types of MAP kinases, Erks and p38, we asked if they were able to compensate for each other. Indeed, we observed that FGF1-dependent Erk1/2 phosphorylation in the presence of p38 inhibitor was slightly stronger (Figure 2a, lanes 8 and 9), whereas in the presence of p38 activator (anisomycin), was notably weaker (Figure 2a, lanes 15 and 16) as compared to the untreated cells (Figure 1a, lane 2). Analysis of signaling kinetics revealed that the inhibition of Erks increased the phosphorylation of p38 (Figure 2a, lanes 23–27 and lanes 30–34).

Next, we verified the impact of p38 inhibition on the activity of Erks without FGF1 stimulation. We found that in the presence of increasing concentrations of the specific p38 kinase inhibitor, SB203580, the basal level of phosphorylated Erk1/2 in serum-starved NIH3T3 was augmented (Figure 3a), showing that reduced activity of one MAP kinase (p38) extorted increased activity of the other (Erk1/2). We hypothesize that this crosstalk functions as a backup system in cells. If one of the two kinases is turned off, the other one can take over.

**Figure 3.** The effect of p38 kinase activity on Erk1/2 activity and FGF1-induced signaling. (**a**) The effect of p38 specific inhibitor on Erk1/2 activity. Serum-starved NIH3T3 cells were treated with increasing concentration of the specific p38 kinase inhibitor SB203580 for 30 min. Then, the cells were lysed and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-Erk1/2 (p-Erk1/2), anti-Erk1/2, and anti-Hsp90 antibodies as a loading control. A representative experiment is shown, n = 4. The graph presents quantification of bands corresponding to phospho-Erk1/2 (p-Erk1/2) normalized to loading control (Hsp90) and expressed as a fold of change in comparison with untreated control. Data are means ± SD of four independent experiments; \*\* *p* < 0.01, \*\*\* *p* < 0.001. (**b**) Schematic representation of synergistic effect of p38 and Erk1/2 in the downregulation of FGF1-induced signaling through FRS2. FGF1-induced tyrosine phosphorylation of FGFR1 leads to the activation of FRS2 followed by GRB2/SOS-mediated activation of RAS and MAP kinases (Erk1/2 and p38). Activated Erks are supported by p38 in phosphorylation of FRS2 (red arrows), constituting a negative feedback loop that results in reduced tyrosine phosphorylation of FRS2 and consequent attenuation of FGFR signaling. Grey dashed line represents functional cross-talk between Erks and p38.

#### **3. Discussion**

Several mechanisms to attenuate FGFR activity have been reported, however, the overall picture of how the FGFR signaling is controlled has not been yet fully described [6]. FRS2 definitely plays an important role in the regulation of FGFR activity, constituting a key component in the Ras/MAPK pathway and being at the same time under control of its effectors (Erks) in a negative feedback loop [17].

Our results show that p38 kinase phosphorylates FRS2 in vitro, and together with Erk1 and Erk2 takes part in modulation of the duration and the intensity of FGFR signaling. Experiments with anisomycin, a p38 activator, revealed that in specific conditions (e.g., stress), p38 can compensate for Erks' action. Despite the similar activity in vitro, the effect of p38 on FRS2 phosphorylation in cells is much weaker than the effect of Erk1 and Erk2. It is probably caused by the fact that growth factors, including FGF1, evoke stronger activation of pro-proliferative MAPKs, i.e., Erks, than stress-response MAPKs, such as p38 kinase [20]. We also found that inhibition of p38 slightly increases the basal activity of Erks. It seems that p38 provides a kind of fine-tuning of the Erks-driven feedback system, taking part in the complicated and multilevel network of downregulation mechanisms of FGFR signaling (Figure 3B). However, to elucidate how one MAP kinase can substitute for another in the absence of FGF ligand, further studies are required. We speculate that inhibition of p38 by SB203580, reducing the degree of FRS2 and FGFR basal serine/threonine phosphorylation, evokes minor upregulation of their tyrosine phosphorylation. A slight increase in FGFR and FRS2 activity results in amplification of Erks phosphorylation.

All MAPK family members share a common phosphorylation site motif [22]. Their substrate specificity is achieved through the docking interactions occurring between the regions outside the phosphorylation site in the substrate and fragments distal from the active site in the kinase [22]. The best characterized docking site involved in interaction with MAPK is the D-site, which consists of a basic region, followed by an LXL motif and a hydrophobic region [22,26]. Interestingly, while Erks require three parts of the D-site, p38 can phosphorylate a substrate without the LXL motif [27]. Another conserved motif, known as the DEF site or the FXFP motif, was identified in several substrates of Erks, including transcription factors, phosphatases, scaffolding, and focal adhesion proteins [28]. It was also shown that p38α has a DEF site motif similar to Erk2, being highly selective for aromatic residues at P1 and P3 position with high preference to Trp [22]. Therefore, it is not surprising that Erk1/2 substrate, FRS2, can also be phosphorylated by p38 kinase.

MAPK signaling seems to be a dynamic map of non-linear interactions rather than static connections. Different MAPK cascades operate in parallel, but at the same time they constitute a network and modulate each other's activity [29]. Here, we have described a cross-talk between two classes of MAPKs in the regulation of FGFR-dependent signaling, but there are other cases of cooperation between different MAPKs. High specificity is observed among upstream kinases, but at the level of MAPK substrate a cross-talk is not unusual [29]. A good example is Elk-1, a member of Ets family of transcription factors controlling the expression of various signaling molecules, which is a target of all three MAPK (Erk1/2, p38, and JNK). Activation of MAPK pathways results in phosphorylation and positive regulation of Elk1, leading to the transcription of early response genes [30]. Another example is MSK1 protein, mitogen-, and stress-activated kinase 1, which is a substrate of both Erks and p38-MAPK during oxidative stress in skeletal myoblasts. Phosphorylation of MSK1 was suggested to influence NF-κB signal specificity in stress response [31]. Another group showed that Erk1/2 and p38 pathways cooperate to promote p21CIP1 expression in order to ensure a sustained G1 cell cycle arrest [32]. The activation of both Erks and p38 was also demonstrated to be essential in regulating delayed STAT3 phosphorylation, as well as in ANF (atrial natriuretic factor) expression profile in response to IL-1β treatment, suggesting their simultaneous role in the development of IL-1β-induced hypertrophy in cardiac myocytes [33]. Furthermore, it was suggested that the function of p38α kinase can be modulated by the cross talk between JNK and p38 kinases [34].

Therapeutic targeting of components of the MAPK cascades is an urgent medical need. Inhibitors of Erks and p38 signaling pathways are in many clinical trials dedicated to treating different types of cancer, inflammation, pain, rheumatoid arthritis, asthma, and neurodegenerative diseases, including Alzheimer's [35,36]. However, it is under question whether targeting of a single signaling molecule can provide an effective therapy. A growing number of drugs are used in combination. Especially in the case of targeting the Erk pathway, vertical inhibition to block two subsequent steps in the cascade is now standard care in a few types of cancer [36,37]. Moreover, to overcome different compensatory mechanisms generated by signaling feedback loops and cross-talks resulting in resistance, efforts are focused to target multiple pathways simultaneously (horizontal inhibition) by combining selective agents [36,38]. Thus, understanding of interplay between different cascades and signaling components is of great importance. Cross-talk between Erk1/2 and p38 and their potential compensation effect should be taken into account during biochemical studies, and might have implications in the design of effective targeted therapies.

### **4. Materials and Methods**

The following primary antibodies were used: rabbit anti-MAPK (Erk1/2, p44/p42; #9102), mouse anti-phospho-MAPK (Erk1/2, p44/p42) (Thr202/Tyr204; #9106), mouse anti-phospho-FGFR (Tyr653/Tyr654; #3476), and rabbit anti-phospho-FRS2α (Tyr196; #3864) from Cell Signaling Technology (Danvers, MA, USA), rabbit anti-FRS2α (H-91; sc-8318), from Santa Cruz Biotechnology (Dallas, TX, USA), mouse anti-phospho-p38 MAPK antibody (Thr180/Tyr182; 612280) and mouse anti-Hsp90 (610418) from BD Transduction Laboratory (San Jose, CA, USA), mouse anti-tubulin (T6557) from Sigma-Aldrich (St Louis, MO, USA). Specific anti-phospho-FGFR1 (Ser777) (pS777-FGFR1) antibody was made by GenScript (Piscataway, NJ, USA) using the following phospho-specific peptide CSMPLDQYpSPSFPDTR. The antibody was purified using the phosphopeptide and by cross-adsorption to the corresponding non-phosphopeptide. HRP-conjugated secondary antibodies were from Jackson Immuno Research Laboratories (West Grove, PA, USA). Heparin-Sepharose CL-6B affinity resin was from GE Healthcare (Piscataway, NJ, USA). Brefeldin A, SB203580, anisomycin, and SL327 were from Calbiochem (San Diego, CA, USA). Heparin, sodium orthovanadate, and U0126 were from Sigma-Aldrich. Phos-tag gels were from Wako Chemicals (Osaka, Japan). All other chemicals were from Sigma-Aldrich.

#### *4.1. Cell Lines and Bacterial Strain*

NIH3T3 cells were grown in DMEM from Thermo Fisher Scientific (Waltham, MA, USA) or (Biowest, Nuaille, France) supplemented with 10% bovine serum (Thermo Fisher Scientific) and 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific). The U2OS cells stably expressing FGFR1 (U2OS-R1) have been described previously [39]. The cells were propagated in DMEM supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin and 0.2 mg/mL geneticin (Invitrogen). For expression of FGF1 wild-type *Escherichia coli* strain Bl21(DE3)pLysS from New England Biolabs (Ipswich, MA, USA) was used.

#### *4.2. Recombinant Proteins*

GST-tagged FRS2 recombinant protein (Q01) was purchased from Anova (Taipei, Taiwan), active human recombinant p38α MAPK was from R&D Systems (Minneapolis, MN, USA) and active human recombinant MAP kinases, Erk1 (p44) and Erk2 (p42), from Calbiochem. Recombinant FGF1 was produced in *E. coli*, as described previously [40].

#### *4.3. Analysis of Signaling Cascades*

Serum-starved cells were treated with 20 ng/mL FGF1 in the presence of 10 U/mL heparin and in the presence or absence of indicated inhibitors for 15 min. Signaling kinetics were carried out in the presence or absence of 2 μg/mL brefeldin A. The cells were lysed with SDS sample buffer, scraped, and sonicated. Total cell lysates were separated by SDS-PAGE or by Phos-tag SDS-PAGE, transferred onto Immobilon-P membrane and subjected to immunoblotting. The sectioned membrane was stripped

with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) and re-probed maximally four times with different antibodies. ImageLab software (version 6.0.1) from Bio-Rad (Hercules, CA, USA) was used to quantify the intensity of bands. The intensity of bands corresponding to phospho-proteins (pY-FGFR, pY-FRS2, p-Erk1/2) was normalized to the intensity of bands corresponding to Hsp90 and then expressed as a fraction of control.
