| 摘要: | 肝細胞生長因子 (hepatocyte growth factor, HGF)，是一個多功能性蛋白質，主要在多種組織的間質細胞及上皮細胞中表現，再生、新生和實質器官的成熟都需要 HGF 參與。在 3D culture， HGF 會促使 MDCK cell 形成腎小管構造。HGF 又稱作 scatter factor，它可以使聚集的細胞離散。Calpain 是 cysteine proteases，為鈣離子依賴型； HGF 會透過調控 calpain，影響與細胞骨架相關蛋白進而調控細胞移動。MST3 為人類 Ste20 蛋白家族的次家族 GCK-III (germinal center kinase family) 的成員，為 serine / threonine kinase，普遍表現在各組織 心、腦、肺、腎、骨骼肌、胎盤組織及細胞中。MST3 已知透過 caspase 切去調控區域，進入核內，參與細胞凋亡。先前研究已知 MST3 透過磷酸化 PTP-PEST，導致與細胞骨架有關蛋白： paxillin 持續磷酸化，抑制細胞移動。但是 MST3 是否參與在 HGF 引起 cell scatter 則並不清楚。我們利用將帶 HA-tagged MST3 及 MST3 kinase dead 的 plasmid 送進 MDCK cell 形成 stable clone。在 HGF 處理下，活性 MST3 參與 HGF 引起的 cell scatter， HGF 活化 calpain，而且切斷 MST3 (MST3 cleavage)，但是沒有活性的 MST3 就不會被切斷。我們用 ALLN 抑制 calpain 活性， MST3 cleavage 就被抑制，也抑制 HGF 引起的 scatter。用 Z-DEVD 抑制 caspase 活性，較無法抑制 HGF 引起的 MST3 cleavage 及 HGF 引起的 scatter。經由上述實驗，我們發現活性 MST3 被 HGF 引起活性的 calpain 切斷，參與 HGF 引起的 cell scatter。
HGF (hepatocyte growth factor, HGF) is a multifunctional protein, mainly in the various tissues of mesenchymal cells and epithelial cells in the performance of regeneration and maturation of organs. In 3D culture, HGF promotes MDCK cell to form a tubular structure. HGF, also known as scatter factor, can make aggregated cells become motile phenotype and scatter. Calpain are calcium-dependent cysteine proteases. HGF regulates cell motility by influence cytoskeletal protein through regulation of calpain. Several mammalian Ste20-like (MST) kinases sharing homology with the yeast ste20 were identified and grouped into two structurally distinct families： p21-activated kinase (PAK) and the germinal center kinase (GCK). MST3, a serine / threonine kinase, belongs to GCK III subfamily. MST3 is widely expressed in the tissue of heart, brain, lung, kidney, skeletal muscle, and placental tissue. Caspase-mediated proteolytic activation of MST3 contributes to apoptosis. Previous studies have shown that MST3 inhibits cell migration in a fashion dependent on autophosphorylation and may regulate paxillin phosphorylation through tyrosine phosphatase PTP-PEST. However, it is not clear whether MST3 is involved in HGF-induced cell scatter. We transfected HA-tag MST3 and MST3 kinase dead plasmid to MDCK cell become stable clone. With HGF treatment, MST3 which has kinase activity is involved in HGF-induced cell scatter. HGF induces calpain activity and MST3 cleavage but MST3 without kinase activity is not cut off. ALLN is a calpain inhibitor. Pretreatment of ALLN results inhibition of calpain activity, MST3 cleavage and HGF induced cell scatter. Pretreatment of Z-DEVD, a caspase inhibitor, doesn't affect the phenomenon of MST3 cleavage and HGF induced cell scatter. By these experiments, we found that with kinase activity of MST3 is cleaved by HGF-activated calpain and cleaved-MST3 is involved in HGF-induced cell scatter. |
