Physiological role of protein-tyrosine phosphatase which regulates the function of hepatocyte growth factor (HGF) receptor

肝细胞生长因子（HGF）是一种多功能的生长因子，可作为多种上皮细胞的有丝分裂原、运动原和形态发生原。HGF的受体是一种跨膜酪氨酸激酶，酪氨酸上的受体磷酸化对于激酶刺激和与细胞内信号转导物的结合都是至关重要的。然而，对HGF受体功能的负调控因子尚不清楚。本课题的目的是鉴定负性调节HGF受体功能的蛋白酪氨酸磷酸酶（Protein-tyrosine phosphatase，PTP 13）。在本课题中，我们取得了以下成果：1.原代培养的大鼠肝细胞裂解液不能水解结构上与磷酸酪氨酸相关的显色分子磷酸对硝基酚，但能快速水解水蛭素C端片段（53-65）的磷酸酪氨酸。因此，我们使用该片段作为底物，鉴定了PTP 3作为原代培养肝细胞中HGF受体的功能性负调节因子. HGF（20 ng/ml）在10 min内可刺激原代培养肝细胞的PTK活性，并呈时间和剂量依赖性，其对PTK活性的刺激作用可被PTK抑制剂钒酸盐完全抑制，但不被Ser/Thr磷酸酶特异性抑制剂冈田酸抑制.在密度依赖性生长停滞的肝细胞中，PTO 2活性升高，表明密度依赖性肝细胞生长抑制涉及原代培养肝细胞中PTO 2活性的调节升高。

Hepatocyte growth factor (HGF) is now known to be a multi-functional growth factor acting as a mitogen, motogen, and morphogen for various types of epithelial cells. The receptor for HGF is a transmenbrane tyrosine kinase and the receptor phosphorylation on tyrosine is critical for both kinase stimulation and association with intracellular signal transducers. However, it is not clear for the negative factor regulating the function of the HGF receptor. The purpose of this project is to identify the protein-tyrosine phosphatase (PTPase) which regulates negatively the function of HGF receptor. In this project, we obtained following results :1. The cell lysates from primary cultured rat hepatocytes had no ability hydrolyse p-nitrophenol phosphate, a chromogenic molecule that is structurally related to phosphotyrosine, but rapidly hydrolysed phosphotyrosine of C-terminal fragment (53-65) of hirudin. Thus, we used this fragment as a substrate to identify the PTPase (s) as a functional negative regulator for HGF receptor in primary cultured hepatocytes.2. HGF (20ng/ml) stimulated the PTPase activity of primary cultured hepatocytes withine 10 min. The effect of HGF on the PTPase activity was time-and dose-dependent.The stimulation of the PTPase activity by HGF was completely inhibited by vanadate, a PTPase inhibitor, but not by okadaic acid, a specific inhibitor for Ser/Thr phosphatase.3. The PTPase activity elevated in density-dependent growth-arrested hepatocytes, suggesting that density-dependent inhibition of hepatocyte growth involves the regulated elevation of the PTPase activity in primary cultured hepatocytes.