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Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

Dominika Czaplinska¹, Lukasz Turczyk², Alicja Grudowska²

¹Department of Molecular Enzymology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Poland; Department of Cell Biology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Poland.

Biochimica Et Biophysica Acta

|July 12, 2014

Abstract:

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.

Insights

p90 ribosomal S6 kinase (RSK) activity is crucial for FGF2/FGFR2 signaling in breast cancer cells. A newly identified FGF2/FGFR2-p38-RSK2 pathway promotes cell proliferation and migration, offering a potential therapeutic target.

Area of Science:

Cell Biology
Molecular Biology
Cancer Research

Background:

p90 ribosomal S6 kinases (RSKs) are key regulators of cell growth and survival, downstream of the MAPK/ERK pathway.
RSKs are implicated in cancer progression, influencing cell motility, invasion, and metastasis.
Fibroblast Growth Factor 2 (FGF2) and its receptor FGFR2 signaling are involved in mammary epithelial and breast cancer cell functions.

Purpose of the Study:

To investigate the role of RSKs in FGF2/FGFR2-driven cellular behaviors in mammary epithelial and breast cancer cells.
To elucidate the upstream regulators of RSK activation within the FGF2/FGFR2 signaling pathway.
To identify potential therapeutic targets for FGFR2-expressing breast tumors.

Main Methods:

Silencing and inhibition of FGFR2 to assess RSK phosphorylation.
Overexpression and stimulation of FGFR2 with FGF2 to evaluate RSK activity.
Treatment with specific kinase inhibitors (ERK, Src, p38) to identify upstream regulators.
Analysis of RSK2 phosphorylation sites (Tyr529, Thr359/Ser363, Thr573, Ser380) using Western blotting.
Assessment of FGF2-mediated anchorage-independent cell proliferation, focal adhesion formation, and cell migration.
Inhibition of RSKs using FMK to evaluate their necessity in FGF2/FGFR2 signaling.

Main Results:

FGFR2 modulation (silencing, inhibition, overexpression, or FGF2 stimulation) directly impacts RSK phosphorylation and activity.
p38 kinase, not MEK/ERK, was identified as an upstream regulator that indirectly activates RSK2 at Tyr529 in FGF2/FGFR2 signaling.
p38-mediated phosphorylation of RSK2-Tyr529 is essential for the activation of other RSK2 residues and downstream signaling in the FGF2/FGFR2 pathway.
FGF2 promotes cell proliferation, focal adhesion formation, and migration, effects abolished by RSK inhibition.
EGF pathway signaling affects RSK2-Tyr529 phosphorylation by p38, but not other RSK sites.

Conclusions:

A novel FGF2/FGFR2-p38-RSK2 signaling pathway is identified, crucial for FGF2/FGFR2-mediated cellular functions.
RSK2 activity is indispensable for FGF2/FGFR2-driven cell proliferation, migration, and focal adhesion formation.
This pathway represents a significant factor in breast cancer pathogenesis and progression, presenting a potential therapeutic target for FGFR2-expressing tumors.

Read full analysis

Keywords:

Breast cancer FGF2 FGFR2 Migration RSK2 Ribosomal S6 kinase

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