Hepatoma-derived growth factor (HDGF) is a novel growth factor originally purified from media conditioned with the human hepatoma cell line HuH-7 Established studies have indicated: HDGF overexpression is related to poor prognosis in various types of cancer including melanoma. HDGF is composed of 240 amino acids and contains two bipartite nuclear location signals (NLSs). To date, the cell cycle distribution and function of HDGF remains largely uncharacterized. By arresting at various cell cycle stages, it was shown that the cellular HDGF level was highest in G1/S phase and lowest in M phase, which was inversely correlated with that of CDK1-CycB kinase. By using in vitro kinase assays, it was found that HDGF is phosphorylated by CDK1, but not glycogen synthase kinase 3b (GSK3b). Bioinformatic search predicted that Ser165 is the putative CDK1 phosphorylation site. Site-directed mutagenesis analysis found that Ser165Ala HDGF mutant was not phosphorylated by CDK1, suggesting Ser165 is indeed the phosphorylation site of CDK1. However, Ser165 mutation had no influence on nuclear targeting of HDGF. In protein stability assay using cyclohexamide, it was found that Ser165Ala mutant HDGF was more stable than the wild type (WT) HDGF. In addition, the HDGF phosphorylation by CDK1 in mitosis resulted in the ubiquitination and degradation of HDGF. Finally, Ser165Ala mutant HDGF was less potent than the WT HDGF in stimulating cell proliferation and migration. Together, these results indicate that CDK1 regulate the stability and function of HDGF.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0827110-182900 |
| Date | 27 August 2010 |
| Creators | Lee, Hsiu-chin |
| Contributors | Yi-ren Hong, Ming-hong Tai, Kuang-hung Cheng |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0827110-182900 |
| Rights | not_available, Copyright information available at source archive |
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