Vigdis Sørensen

Different intracellular trafficking of FGF1 endocytosed by the four homologous FGF receptors.

Many growth factors and cytokines bind to more than one receptor, but in many cases the different roles of the separate receptors in signal transduction are unclear. Intracellular sorting of ligand-receptor complexes may modulate the signalling, and we have here studied the intracellular trafficking of ligand bound to receptors for fibroblast growth factors (FGFs). For this purpose, we transfected HeLa cells with any one of the four tyrosine kinase FGF receptors (FGFR1-4). In cells expressing any one of these receptors, externally added FGF1 was localized to sorting/early endosomes after 15 minutes at 37°C. After longer incubation times, FGF1 internalized in cells expressing FGFR1 was localized mainly to late endosomes/lysosomes, similarly to EGF. By contrast, FGF1 internalized in cells expressing FGFR4 followed largely the same intracellular pathway as the recycling ligand, transferrin. In cells expressing FGFR2 or FGFR3, sorting of FGF1 to lysosomes was somewhat less efficient than that observed for FGFR1. Furthermore, FGF1 was more slowly degraded in cells expressing FGFR4 than in cells expressing FGFR1-3 and in addition, internalized FGFR4 as such was more slowly degraded than the other receptors. The data indicate that after endocytosis, FGFR4 and its bound ligand are sorted mainly to the recycling compartment, whereas FGFR1-3 with ligand are sorted mainly to degradation in the lysosomes. Alignment of the amino acid sequence of the intracellular part of the four FGFRs revealed several lysines conserved in FGFR1-3 but absent in FGFR4. Lysines are potential ubiquitylation sites and could thus target a receptor to lysosomes for degradation. Indeed, we found that FGFR4 is less ubiquitylated than FGFR1, which could be the reason for the different sorting of the receptors.

Phosphorylation of Fibroblast Growth Factor (FGF) Receptor 1 at Ser777 by p38 Mitogen-Activated Protein Kinase Regulates Translocation of Exogenous FGF1 to the Cytosol and Nucleus▿

ABSTRACT Exogenous fibroblast growth factor 1 (FGF1) signals through activation of transmembrane FGF receptors (FGFRs) but may also regulate cellular processes after translocation to the cytosol and nucleus of target cells. Translocation of FGF1 occurs across the limiting membrane of intracellular vesicles and is a regulated process that depends on the C-terminal tail of the FGFR. Here, we report that translocation of FGF1 requires activity of the α isoform of p38 mitogen-activated protein kinase (MAPK). FGF1 translocation was inhibited after chemical inhibition of p38 MAPK or after small interfering RNA knockdown of p38α. Translocation was increased after stimulation of p38 MAPK with anisomycin, mannitol, or H2O2. The activity level of p38 MAPK was not found to affect endocytosis or intracellular sorting of FGF1/FGFR1. Instead, we found that p38 MAPK regulates FGF1 translocation by phosphorylation of FGFR1 at Ser777. The FGFR1 mutation S777A abolished FGF1 translocation, while phospho-mimetic mutations of Ser777 to Asp or Glu allowed translocation to take place and bypassed the requirement for active p38 MAPK. Ser777 in FGFR1 was directly phosphorylated by p38α in a cell-free system. These data demonstrate a crucial role for p38α MAPK in the regulated translocation of exogenous FGF1 into the cytosol/nucleus, and they reveal a specific role for p38α MAPK-mediated serine phosphorylation of FGFR1.

Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail.

Members of the fibroblast growth factor family bind to one or more of the four closely related membrane-spanning FGF receptors. In addition to signaling through the receptors, exogenous FGF-1 and FGF-2 are endocytosed and translocated to the cytosol and nucleus where they stimulate RNA and DNA synthesis. Here we have studied the ability of the four FGF receptors to facilitate translocation of exogenous FGF-1 to the cytosol and nucleus. FGFR1 and FGFR4 were able to mediate translocation, whereas FGFR2 and FGFR3 completely lacked this ability. By analyzing mutant FGFRs we found that the tyrosine kinase domain could be deleted from FGFR1 without abolishing translocation, whereas the C-terminal tail of the FGFRs, constituted by approximately 50 amino acids downstream of the kinase domain, plays a crucial role in FGF-1 translocation. Three amino acids residues within the C-terminal tail were found to be of particular importance for translocation. For FGFR2, the two amino acid substitutions Q774M and P800H were sufficient to enable the receptor to support FGF-1 translocation. The results demonstrate a striking diversity in function of the four FGFRs determined by their C-terminal domain.

Nuclear Import of Exogenous FGF1 Requires the ER‐Protein LRRC59 and the Importins Kpnα1 and Kpnβ1

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine‐rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA‐mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin‐α1 (Kpnα1), and Kpnβ1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnβ1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane‐anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS‐like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnβ1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.

A Nuclear Export Sequence Located on a β-Strand in Fibroblast Growth Factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C δ induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a β-strand, which has not been reported before, since NESs usually are α-helical.

Deletion mutant of FGFR4 induces onion-like membrane structures in the nucleus

The expression of several deletion mutants of fibroblast growth factor receptor 4 (FGFR4) was studied in COS-1 cells. FGFR4-mutants lacking most of the extracellular region did not efficiently reach the plasma membrane but accumulated in the endoplasmic reticulum (ER) and Golgi body. A mutant FGFR4 lacking the kinase domain as well as most of the extracellular region (ΔExt/R4Tth) had a distinct intracellular distribution. It localized in part to the nucleus, where it exhibited a striking spotted pattern. Ultrastructural studies showed that the nuclear spots consisted of several layers of membrane that were folded into onion-like structures at the nucleoplasmic side of the nuclear envelope. These intranuclear structures did not contain nuclear pores but were positive for the ER proteins calreticulin and protein disulfide isomerase, in addition to abundant ΔExt/R4Tth. Formation of the intranuclear structures was sensitive to inhibition of protein kinase C. Live microscopy of a green-fluorescent-protein/ΔExt/R4Tth fusion protein showed that the intranuclear structures were stable and immobile, suggesting that they function as deposits of the overexpressed mutant and associated membrane. The ΔExt/R4Tth protein also induced formation of densely packed membrane stacks in the cytosol and we suggest a model were the intranuclear structures are formed by invagination of ER-derived membrane stacks into the nucleus.

Nucleolin Regulates Phosphorylation and Nuclear Export of Fibroblast Growth Factor 1 (FGF1)

Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1.

Fibroblast growth factor receptor-induced phosphorylation of STAT1 at the Golgi apparatus without translocation to the nucleus.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines, but little is known about activation of STAT1 from intracellular sites. Here we show that transient transfection of COS cells with fibroblast growth factor receptors (FGFRs) led to ligand‐independent phosphorylation of the receptors, including intracellular immature forms. FGF‐independent activation of STAT1 was demonstrated at the Golgi apparatus where it was colocalized with FGFRs. Both FGFR1 and FGFR2 induced strong phosphorylation of STAT1 causing redistribution of the Golgi apparatus, while FGFR3 and FGFR4 induced less phosphorylation of STAT1 and little or no redistribution of the Golgi apparatus. Upon expression of a cytosolic mutant of FGFR4 lacking the transmembrane as well as the extracellular region (CytR4), STAT1 was phosphorylated and transferred to the nucleus. The results indicate that immature forms of FGFRs form incomplete signaling complexes on Golgi membranes trapping phospho‐STAT1 on this organelle. J. Cell. Physiol. 212: 148–156, 2007.

Translocation of exogenous FGF1 into cytosol and nucleus is a periodic event independent of receptor kinase activity

Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~6 h, ~30 h, and ~54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.

Cytotoxic Conjugates of Fibroblast Growth Factor 2 (FGF2) with Monomethyl Auristatin E for Effective Killing of Cells Expressing FGF Receptors

Antibody–drug conjugates (ADCs) are a new class of anticancer therapeutics that combine the selectivity of targeted treatment, ensured by monoclonal antibodies, with the potency of the cytotoxic agent. Here, we applied an analogous approach, but instead of an antibody, we used fibroblast growth factor 2 (FGF2). FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a cell-surface receptor reported to be overexpressed in several types of tumors. We developed and characterized FGF2 conjugates containing a defined number of molecules of highly cytotoxic drug monomethyl auristatin E (MMAE). These conjugates effectively targeted FGFR1-expressing cells, were internalized upon FGFR1-mediated endocytosis, and, in consequence, revealed high cytotoxicity, which was clearly related to the FGFR1 expression level. Among the conjugates tested, the most potent was that bearing three MMAE molecules, showing that the cytotoxicity of protein–drug conjugates in vitro is directly dependent on drug loading.