Abstract
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 delta 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 beta-strand, which has not been reported before, since NESs usually are alpha-helical.
Highlights
Fibroblast growth factor-1 (FGF-1)5 is a member of a superfamily of growth factors involved in numerous cellular processes, such as differentiation, proliferation, and migration [1]
Characterization of the Putative Nuclear Export Sequence by Amino Acid Substitutions—Upon visual examination of the amino acid sequence of FGF-1, we noticed a cluster of hydrophobic amino acids at the C terminus (144ILFLPLPVSSD-Stop) that resembles the loose consensus of Leu-rich nuclear export sequence (NES)
To study the transport of exog- the phosphorylated mutants were only found in the nuclear enous FGF-1 to the cytosol and nucleus of cells, we have estab- fraction (Fig. 1A, lanes 2 and 3)
Summary
Fibroblast growth factor-1 (FGF-1)5 is a member of a superfamily of growth factors involved in numerous cellular processes, such as differentiation, proliferation, and migration [1]. To study the transport (translocation) of exog- the phosphorylated mutants were only found in the nuclear enous FGF-1 to the cytosol and nucleus of cells, we have estab- fraction (Fig. 1A, lanes 2 and 3). FGF-1 was detected in the nucleus when the nuclear export was FGF-1 possesses a single phosphorylation site, Ser130, which inhibited by LMB (Fig. 1A, lane 4).
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