肝癌衍生誘導生長因子(hepatoma-derived growth factor; HDGF)，為近年來發現的生長因子，由人類肝癌細胞株 Huh-7的培養液中所純化出，日前已有許多研究指出: HDGF的過度表現與許多的癌症患者的癒後相關連，其中也包括了黑色素癌。HDGF由240個胺基酸所組成，包含兩個核定位訊息序列(NLS),至今HDGF在細胞週期中的分佈以及功能大多未明瞭，將細胞同步化後，發現HDGF在進入細胞週期的S期前表現最高並隨著細胞週期的進行降低表現直到細胞進行有絲分裂，相對地，CDK1-CycB激酶的活性隨之上升，利用生物體外激脢活性試驗 (in vitro kinase assay)，證實了CDK1會磷酸化位於第二個核定位訊息序列上的Ser165，然而，將165位上的Ser進行點突變並不影響HDGF的進核能力，此外，利用cyclohexmide進行蛋白質穩定度實驗發現CDK1的磷酸化會影響HDGF的穩定度，且經由UPS (ubiquitin-proteasome system)將HDGF降解, 功能性研究上Ser165的突變改變了HDGF促細胞生長及移動能力，總結的說Ser165這個胺基酸的磷酸化對於蛋白質穩定度、刺激細胞生長及移動能力是很重要的。

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.

Abstract in Chinese 04
Abstract in English 05
Abbreviations 06
Introduction 07
Specific Aims 13
Materials and Methods 15
Results 23
Discussion 28
Table 31
Figures 32
Appendix 47
References 53

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