Abstract
Members of high (22-, 22.5-, 24-, and 34-kDa) and low (18-kDa) molecular mass forms of fibroblast growth factor-2 (FGF-2) regulate cell proliferation, differentiation, and migration. FGF-2s have been previously shown to accumulate in the nucleus and nucleolus. Although high molecular weight forms of FGF-2 contain at least one nuclear localization signal (NLS) in their N-terminal extension, the 18-kDa FGF-2 does not contain a standard NLS. To determine signals controlling the nuclear and subnuclear localization of the 18-kDa FGF-2, its full-length cDNA was fused to that of green fluorescent protein (GFP). The fusion protein was primarily localized to the nucleus of COS-7 and HeLa cells and accumulated in the nucleolus. The subcellular distribution was confirmed using wild type FGF-2 and FGF-2 tagged with a FLAG epitope. A 17-amino acid sequence containing two groups of basic amino acid residues separated by eight amino acid residues directed GFP and a GFP dimer into the nucleus. We systematically mutated the basic amino acid residues in this nonclassical NLS and determined the effect on nuclear and nucleolar accumulation of 18-kDa FGF-2. Lys(119) and Arg(129) are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys(128) regulates only nucleolar localization of 18-kDa FGF-2. Together, these results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite NLS, a portion of which also regulates its nucleolar localization.
Highlights
Members of high (22, 22.5, 24, and 34-kDa) and low (18-kDa) molecular mass forms of fibroblast growth factor-2 (FGF-2) regulate cell proliferation, differentiation, and migration
Lys119 and Arg129 are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys128 regulates only nucleolar localization of 18-kDa FGF-2. These results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite nuclear localization signal (NLS), a portion of which regulates its nucleolar localization
These results indicate that the 17-mer containing the NLS of 18-kDa FGF-2 can direct green fluorescent protein (GFP) and the GFP dimer into the nucleus but not as efficiently as full-length FGF-2
Summary
Plasmid Construction—To construct pEGFP-N1-FGF-2 encoding the fusion protein GFP-FGF-2, the full-length cDNA of human 18-kDa FGF-2 The amplified DNA was digested with BsrGI and NotI and ligated to the corresponding sites at the 3Ј-end of GFP cDNA in pEGFP-N1 (Clontech). The full-length cDNA of FGF-2 was placed at the 5Ј-end of GFP cDNA by first amplifying it using forward (5Ј-TTTAAGCTTATGGCAGCCGGGAGCATCACC-3Ј) and reverse (5Ј-TTTCTGCAGGCTCTTAGCAGACATTGGAAG-3Ј) primers containing HindIII and PstI sites, respectively. To generate pNLS-dEGFP-N1, in which the NLS of 18-kDa FGF-2 is fused to a GFP dimer, GFP cDNA was amplified with primers containing BsrGI sites. Site-directed Mutagenesis—To obtain a plasmid encoding wild type 18-kDa FGF-2, two stop codons were introduced between FGF-2 and GFP cDNA in pFGF-2-EGFP-N1 using the QuikChange® site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instruction. Mutations K128G and R129G were introduced into the mutant R116G/R118G to generate R116G/R118G/K128G and R116G/R118G/R129G, respectively, using the corresponding primers described above. The secondary antibody was detected using SuperSignal chemiluminescent substrate (Pierce) according to the manufacturer’s instructions
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