肝癌衍生生長因子在許多種細胞中是位細胞核內刺激生長，此外在肝癌和肺癌的癌細胞的形成及病人的存活率中，與肝癌衍生生長因子的過度表現有關。 肝癌衍生生長因子(hepatoma-derived growth factor; HDGF)是由240個氨基酸所組成，具有2個nuclear localization signals (NLSs)。 我們發現HDGF 之PWWP (HDGF 1-100氨基酸) 與 C-140 (101-240氨基酸) domain分別具有一個NLS所以皆會進入核內。 但C-140 具HDGF促進細胞生長和移行之功能，但是PWWP卻沒有這樣的性質。 NLS突變會阻斷肝癌衍生因子進入細胞核和促進生長的能力，但卻不影響它促進細胞移行的能力。此外Ser165位於第二個NLS2裡，被預測為cdc2磷酸化的位置。以in vitro磷酸激

Hepatoma-derived growth factor (HDGF) is a nucleus-targeting mitogen for various types of cells. Besides, HDGF overexpression is associated with tumor progression and poor survival outcome in patients with hepatocellular carcinoma (HCC) and lung cancer. HDGF is capable of promoting the proliferation and migration in various types of cells. HDGF is composed of 240 amino acids and contains 2 putative bipartite nuclear localization signals (NLSs). By dividing HDGF into two deletion domains: PWWP (residues 1-100) and C140 (residues 101-240), we found that both PWWP and C140 domains are capable of promoting nuclear localization, However, only C140 domain promoted cell proliferation and migration as HDGF. Mutation in NLS domains abrogated the nuclear localization and growth-promoting function, but not the migratory potential of HDGF. Beside, Ser165 was predicted as putative cdc2 phosphorylation site. In vitro kinase assay indicated that Ser165 of HDGF is the phosphorylated site of cdc2 kinase. We also demonstrated that mutations in cdc2 phoshprylation site did not affect the nuclear localization, proliferation-stimulating activities of HDGF but enhance migration-stimulating abilities of HDGF. Recently, the HDGF domain containing residues 81 to 100 is shown to be responsible for binding to membrane receptor in NIH3T3 cells. Besides, Lys96 plays a pivotal role for receptor binding. By generation of HDGF Lys96A mutant protein, we found that mutation of Lys96 indeed caused a prominent reduction in cellular binding affinity of HDGF to NIH3T3 cells and affect cell migration. In summary, the NLSs are essential for the mitogenic effect of HDGF, but not required for migration. And the cdc2 phoshorylation site is important for NIH3T3 migration. The Lys96 of HDGF play an important role of membrane receptor binding and cell migration.

Index
Abstract in Chinese……………………………………………………….I

Abstract in English……………………………………………………….II


contents
Introduction………………………………………………………………1

Specific Aims…………………………………………………………….7

Materials and Methods…………………………………………………...8

Results…………………………………………………………………..18

Discussion……………………………………………………………….23

Refference……………………………………………………………….26

Table…………………………………………………………………….30

Figures…………………………………………………………………33

Appendix………………………………………………………………..52

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