Abstract
Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.
Highlights
Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence and nuclear localization signals (NLSs) in gene-specific regions other than the hath region
To clarify whether HDGF can translocate to the nucleus and which NLS is the functional sequence in the HDGF molecule, 293 cells were transfected with an expression vector encoding a green fluorescence protein (GFP)-tagged human HDGF cDNA and mutant HDGF cDNAs
In the transfected cells expressing GFP-HDGFmN2 or the truncated form without NLS2 (GFP-HDGF-SA), green fluorescence was detected in the nucleus, with some amount found in the cytoplasm
Summary
Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. HDGF family proteins have a basic motif homologous to the reported consensus sequences for bipartite nuclear localization signals (NLSs) in gene-specific amino acid sequences other than the hath region of the molecule [1, 2, 6, 7]. Cytokines, and hormones identified previously, the following polypeptide factors have been reported to contain one or more NLSs and to undergo nuclear translocation for mitogenesis: acidic or basic fibroblast growth factor (FGF), FGF-3, schwannoma-derived growth factor (SDGF), ciliary neurotrophic factor, amphiregulin, angiogenin, and lens epithelium-derived growth factor (LEDGF) (8 –23) These findings prompted us to study whether HDGF translocates to the nucleus via these NLSs, and whether its nuclear translocation is essential for induction of cell growth activity. We analyze which part of the HDGF molecule is essential for internalization and the mitogenic activity
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