Exogenously Added Fibroblast Growth Factor 2 (FGF-2) to NIH3T3 CellsInteracts with Nuclear Ribosomal S6 Kinase 2 (RSK2) in a Cell Cycle-dependentManner

Stéphane Roga,Karine Bailly,Fabienne Soulet,François Amalric,Anne-Claire Lavigne,Gérard Bouche

doi:10.1074/jbc.m500232200

Stéphane Roga, Karine Bailly + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m500232200

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Abstract

Fibroblast growth factor 2 (FGF-2) has been detected in the nuclei of many tissues and cell lines. Here we demonstrate that FGF-2 added exogenously to NIH3T3 cells enters the nucleus and interacts with the nuclear active 90-kDa ribosomal S6 kinase 2 (RSK2) in a cell cycle-dependent manner. By using purified proteins, FGF-2 is shown to directly interact through two separate domains with two RSK2 domains on both sides of the hydrophobic motif, namely the NH2-terminal kinase domain (residues 360-381) by amino acid Ser-117 and the COOH-terminal kinase domain (residues 388-400) by amino acids Leu-127 and Lys-128. Moreover, this interaction leads to maintenance of the sustained activation of RSK2 in G1 phase of the cell cycle. FGF-2 mutants (FGF-2 S117A, FGF-2 L127A, and FGF-2 K128A) that fail to interact in vitro with RSK2 fail to maintain a sustained RSK2 activity in vivo.

Highlights

Fibroblast growth factor 2 (FGF-2)1 is a member of the large FGF family consisting of 30 members in humans [1]
Several of the newly phosphorylated proteins are known to be casein kinase 2 (CK2) substrates [19, 20]. In agreement with this result, we have shown a direct interaction between CK2 and nuclear FGF-2 in vitro by incubation of a nuclear extract with Biotinylated FGF-2 (B-FGF-2) immobilized on streptavidin beads [18] and in vivo by coimmunoprecipitation of CK2 and FGF-2 from NIH3T3 cells undergoing G0/S transition [13]
To identify the kinase activities able to interact with BFGF-2 and phosphorylate H3, we incubated a nuclear extract from NIH3T3 exponential growing cells with B-FGF2 immobilized on streptavidin beads and eluted bound proteins at 0.4 and 0.5 M NaCl and performed a kinase assay

Summary

Introduction

Fibroblast growth factor 2 (FGF-2) is a member of the large FGF family consisting of 30 members in humans [1] It is involved in various cellular processes such as stimulation of DNA synthesis and cell proliferation, as well as differentiation and cell migration. FGF-2 signaling through both FGF receptors and nuclear targets is required for the stimulation of cell proliferation [13, 14]. These data show that nuclear localization is a general phenomenon for some growth factors, suggesting nuclear functions independent of the functions as extracellular factors. The interaction of nuclear FGF-2 with pluripotent RSK2 offers a new mechanism through which FGF-2 may control fundamental cellular processes

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