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

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 NH 2 -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 G 1 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. Fibroblast growth factor 2 (FGF-2)1 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. In vitro, numerous cell types synthesize FGF-2 in five molecular isoforms. Four of these isoforms have high molecular masses (21.5,22,24, and 34 kDa) and are initiated at alternative CUG codons. The main form (18 kDa) is initiated at a regular AUG codon. The high molecular mass forms localize exclusively in the nucleus, their NH 2 -terminal extension containing a nuclear localization sequence (2, 3). The 18-kDa isoform is primarily cytoplasmic and, despite the lack of a classical signal peptide, is released by a mechanism bypassing the classical export route taken by most secreted proteins via the endoplasmic reticulum and the Golgi apparatus (4).The pleiotropic effects exhibited by 18-kDa FGF-2 reflect an intricate combinatorial process involving interactions between the growth factor, any of four closely related high affinity transmembrane tyrosine kinase receptors (FGFR1-4), and low affinity binding sites corresponding to the naturally heterogeneous glycosaminoglycan chains of heparan sulfate proteoglycans. The interaction of FGF-2 with its receptor induces a phosphorylation cascade that results in the activation of signalization pathways. Furthermore, in addition to interactions with cell surface receptors, several growth factors enter the nucleus of target cells, either alone or associated with their receptors (5-9). Nuclear translocation of internalized FGF-1 and FGF-2 is an essential step in their mitogenic activity (10 -12). 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.For the understanding of the nuclear functions of FGF-2, identification of interacting proteins is a crucial step. Here, we report that the exogenously added...

Heterodimerization of FGF-receptor 1 and PDGF-receptor-α: a novel mechanism underlying the inhibitory effect of PDGF-BB on FGF-2 in human cells

“…We then investigated FGF-2 internalization, an event that follows binding to and activation of FGF-R1 and leads FGF-2 to the nucleus, triggering the expression of genes regulating cell cycle and cellular growth. 26 As for FGF-R1 phosphorylation, FGF-2 internalization was strongly reduced in the presence of PDGF-BB ( Figure 3B). Thus we hypothesized that PDGF-BB may act through a mechanism likely mediated by its receptors.…”

Functional diversity of FGF‐2 isoforms by intracellular sorting

“…Mutants that fail to interact with RSK2, but not with FGFRs, were around 50% less mitogenic than wild type of FGF-2, and were not able, similarly to EGF or FCS, to maintain the RSK2 in an active form. (92) These observations indicate that exogenous FGF-2 that is translocated to the nuclear compartment is able to interact with different kinases and regulate their activity, and this is reflected in the cellular/mitogenic response to the growth factor.…”

“…Table 1 gives an overview of studies (20) NIH/3T3 18 kDa, 22 kDa, 22.5 kDa, 24 kDa Serum independent growth (50,56,57) NIH/3T3 18 kDa, hmw Altered growth and morphology (100) NIH/3T3 and A31 18 kDa, hmw Serum independent growth, transformed morphology, altered drug resistance (101) Cardiac myocytes Hmw Proliferation, binucleation, chromatin compaction (59,67) or mitotic arrest, chromatin disruption (68) Astrocytes and glioma cells 18 kDa, 22 kDa, 22.5 kDa, 24 kDa Proliferation (60) AR4-2J Hmw Proliferation (61,65,102) , differntial expression of PKC d and e, and ERK-1/2 activation (61,65) and c-Jun expression (61) HeLa Hmw Induction of a radioresistant phenotype (63) Sympathetic neurons 23 kDa Induction of a multinuclear phenotype (69) Bladder carcinoma cells 24 kDa Formation of metastasis (71,72) Schwann cell precursors Hmw Transdifferentiation into melanocytes (62) Schwann and PC12 cells 18 kDa, hmw Altered growth and differential regulation of neuronal or endocrine phenotype (103,104) Schwann cells 21 kDa, 23 kDa Sensory nerve cell recovery and axon myelination (70) Schwann cells 23 kDa Interaction with SMN (a factor in spliceosomal complexes) (52,78) SK-Hep1 18 kDa, hmw Transformation, tumorigenicity (105) SK-Hep-1 Hmw Interaction with antiapoptotic protein FIF (77) ABAE Hmw Immortalization (106) NIH/3T3 Hmw Binding to ribosomal protein L6/TAXREB107 (76) (In vitro) 18 kDa, 23 kDa Interaction with splicing factor SF3a66 (79) NIH/3T3 Exogenous 18 kDa Mitogenesis/Interaction with and stimulation of CK2 (90) NIH/3T3 Exogenous 18 kDa Mitogenesis/Interaction with and activation of ribosomal S6 kinase 2 (92)…”

“…(90)(91)(92) This trait of FGF-2 is shared with FGF-1; several studies have demonstrated the occurrence of translocation of exogenous FGF-1 to cytosol and nucleus and its role in induction of mitogenesis (See Refs. 13,21 for review).…”

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines

“…Interestingly, the growth factors SDGF [33], HDGF [34,35], and FGF-1 [36,61] need to be translocated to the nucleus in order to be mitogenically active. Furthermore, the nuclear presence of the growth factors FGF-2, LEDGF and SDGF play roles in transcriptional regulation: Nuclear FGF-2 binds and activates the kinases RSK2 and CKII [62,63], nuclear LEDGF/p75 binds to heat shock elements and stress-related regulatory elements in promoters of many stress-related genes and thereby activates their expression [64], and SDGF binds AT-rich elements in promoters of the immediate early genes NGFI-A and c-fos and induces their expression [33]. Based on our observations that PDGF-C is present in the nucleus of human thyrocytes in tissue and in cultured cells (Figs.…”