**1. Introduction**

Fibroblast growth factor (FGF) receptor (FGFR) -dependent signaling plays a crucial role during embryonic development, as well as in adult life. It stimulates growth, differentiation, survival, injury repair, regeneration, and metabolism. Consequently, excessive activation of FGFRs may result in severe abnormalities, such as cancer development and progression, and skeletal disorders [1].

Downregulation of FGFR signaling consists of a number of mechanisms that enable precise control of cellular outcome. They include regulation of FGFR synthesis, receptor internalization, followed by degradation and modulation of FGFR tyrosine phosphorylation via the activity of several negative regulators and feedbacks [1–6].

Previously we showed that phosphorylation of Ser777 of FGFR1 by activated MAPK Erk1 and Erk2 significantly reduces the tyrosine phosphorylation in the kinase domain of the receptor. We suggested that the phosphorylated Ser777 could act as a binding site for tyrosine phosphatases responsible for receptor inactivation [7]. FGFR1 and FGFR2 are also phosphorylated on Ser779 in response to FGF. Upon phosphorylation, this residue serves as a binding site for 14-3-3 family of

phosphoserine/threonine-binding adaptor/scaffold proteins and tunes up Ras/MAPK signaling [8,9]. In addition, ribosomal S6 kinase 2 (RSK2) phosphorylates Ser789 of FGFR1. Inhibition of RSK2 activity leads to prolonged tyrosine phosphorylation of FGFR1 resulting from reduced FGFR1 ubiquitination and endocytosis [10].

Furthermore, FGFR downstream signaling molecules are also affected by inhibitory loops and negative regulators, including Sprouty proteins (SPRY1–SPRY4), MAPK phosphatase 3 (MKP3), SEF (similar expression to FGF), and protein tyrosine phosphatase receptor type G (PTPRG). MKP3 directly dephosphorylates MAPK turning off the Ras cascade [11]. Sprouty is phosphorylated in response to FGFR activation and competes for Grb2 (growth factor receptor-bound protein 2), binding to FRS2 and Shp2 (SH2 domain-containing protein tyrosine phosphatase-2), preventing Ras activation. Phosphorylated Sprouty also binds to Raf and blocks subsequent MAPK signaling [12,13]. SEF protein, in addition to its role in inhibiting MAPK signaling, is capable of interacting directly with FGFRs and blocking receptor phosphorylation [14,15]. The protein tyrosine phosphatase receptor type G, PTPRG, directly dephosphorylates the receptor itself, thereby turning off the signaling [16].

However, one of the most effective downregulation mechanism described so far is based on Erk1/2-FRS2 negative feedback loop [17]. FRS2, a lipid anchored adaptor protein, is associated constitutively through its PTB domain with the juxtamembrane domain of FGFR and is phosphorylated on multiple tyrosines upon receptor activation [18,19]. Phosphorylated FRS2 forms two specific binding sites for Shp2 and four binding sites for Grb2. Grb2 is constitutively bound via its SH3 domains to Sos and Gab1, and these proteins constitute a signaling complex activating the RAS/MAPK and PI3K/Akt pathways [1]. Activated extracellular signal-regulated protein kinases (Erk1/2), irrespective of the upstream receptor tyrosine kinase (RTK), phosphorylate FRS2 at eight threonines (PXTP motifs) [17]. Such phosphorylation reduces FRS2 tyrosine phosphorylation, decreases recruitment of Grb2, and attenuates downstream signaling response, providing a control mechanism to regulate FGFR activity [17].

Besides Erks, the MAPK family includes also p38 and c-Jun N-terminal kinases (JNKs). Erks are activated by mitogenic factors and are associated with growth, differentiation, and proliferation, whereas p38 and JNKs take part mainly in the cell response to stress conditions and inflammatory cytokines, and are activated only weakly by proliferative stimuli [20]. There are four isoforms of p38 identified so far in mammals, among which p38α and p38β are the best characterized and most widely expressed [21].

Interestingly, all MAPKs share specificity for a common consensus phosphorylation site [22]. We showed previously that two different MAPKs, Erk1/2 and p38, are able to phosphorylate FGFR1 at the same serine residue [7,23]. Phosphorylation of FGFR1 by p38 seems to be necessary for FGF1 to be translocated into the cytosol and nucleus upon internalization [23], while phosphorylation by Erk1/2 provides a negative-feedback mechanism that controls FGF signaling, and thereby protects the cell against excessive activation of FGFR [7].

Here, we found that in addition to Erks, p38 can also phosphorylate FRS2 and modulate its phosphorylation at tyrosine residues. Simultaneous inhibition of both types of MAPKs resulted in prolonged activation of FGFR1, FRS2, and downstream signaling, as compared to the elimination of Erks activity alone. Our results reveal a novel regulatory feedback mechanism, where p38 is able to partially substitute Erk1 and Erk2 in the regulation of FGFR activity.

#### **2. Results**

#### *2.1. The p38 Activity Influences Electrophoretic Mobility Shift of FRS2*

We analyzed by immunoblotting the activation of FGF1-induced signaling in NIH3T3 mouse fibroblast cells upon 15-min stimulation with FGF1 in the presence of different combinations of MAP kinases inhibitors (MEK1/2 inhibitors, p38 inhibitor) and p38 kinase activator (anisomycin) (Figure 1a). As expected, in the presence of specific MEK1/2 inhibitors (U0126 or SL327, lanes 3, 4, 5, 6) that prevent

Erk1/2 activation, we observed augmented tyrosine phosphorylation of FGFR1 (Tyr653/Tyr654) and lack of phosphorylation at Ser777. In addition, MEK1/2 inhibition led to increased tyrosine phosphorylation of FRS2. This is in accordance with findings by Lax and co-workers [17]. The band for FRS2, visualized by antibodies recognizing total and phoshorylated (Tyr196) forms, displayed faster migration (lanes 3 and 4) as compared to the cells untreated with inhibitor (lane 2). Interestingly, we also found that in the simultaneous presence of MEK1/2 inhibitor (U0126) and p38 inhibitor (SB203580) (lane 5), the FRS2 band migrated slightly faster than in the presence of U0126 or SL327 alone (lanes 3 and 4). Addition of anisomycin (p38 activator) in the presence of U0126 resulted in a small up-shift of the FRS2 band (lane 6). We confirmed these findings using another model cell line, human osteosarcoma U2OS cells that stably expresses FGFR1 (U2OS-R1) (Figure 1b).

**Figure 1.** The effect of p38 activity on FRS2. (**a**–**c**) FGF1-induced electrophoretic mobility shift of FRS2. Serum-starved (**a**,**c**) NIH3T3 and (**b**) U2OS-R1 were pretreated for 20 min with or without MEK1/2 inhibitors (20 μM U0126, 1 μM SL327), p38 inhibitor (5 μM SB203580), and p38 activator (10 μM anisomycin), and then stimulated with the growth factor in the presence of heparin (10 U/mL) for 15 min. Cells were lysed, and the cellular material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (**a**,**b**) or Phos-Tag SDS-PAGE (**c**) 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 or anti-tubulin as a loading control. (**d**) In vitro phosphorylation of FRS2 by p38α kinase. Recombinant, active kinase p38α and partial recombinant fusion protein FRS2α with GST tag were incubated with (γ-33P) ATP in reaction buffer at 30 ◦C for 30 min in the presence or absence of 5 μM SB203580. Erk1 and Erk2 kinases served as positive controls. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). Representative experiments are shown, for (**a**) and (**b**) n = 4, and for (**c**) and (**d**) n = 2.

As conventional SDS-PAGE is not optimal for separation of phosphorylated populations of a protein from a non-modified fraction, we studied FRS2 migration upon treatment with various kinases inhibitors using a Phos-Tag technology that is well suited for phosphorylation profiling of proteins [24,25]. Cell lysates were separated on Phos-Tag gels followed by immunoblotting with anti-FRS2 antibody (Figure 1c).

In the serum starved NIH3T3 cells, FRS2 was observed mainly as a single, fast migrating band on Phos-Tag gel, likely representing non-phosphorylated FRS2 (Figure 1c, lane 1). Stimulation of cells with FGF1 drastically changed the migration pattern of FRS2 in the Phos-Tag gel. FRS2 was detected in three slowly migrating bands that represent differentially phosphorylated FRS2 (Figure 1c, lane 2). Treatment of cells with MEK1/2 inhibitor caused clear changes in the phosphorylation status of FRS2 (Figure 1c, lane 3). Interestingly, concomitant inhibition of p38 and MEK1/2 altered gel migration of FRS2 in relation to the inhibition of either MEK1/2 or p38 alone (Figure 1c, lane 5 vs. lanes 3 and 4). In agreement with these findings, the over-activation of p38 by anisomycin, either in the presence or absence of MEK1/2 inhibitor, altered the migration of FRS2 on Phos-Tag gels (Figure 1c, lanes 6 and 7 vs lanes 2 and 3). These data suggest that p38 kinase affects the phosphorylation state of FRS2.
