Role and regulation of phosphatidylinositol 3-kinase in melanoma cells

磷脂酰肌醇3-激酶在黑色素瘤细胞中的作用和调节

基本信息

批准号：
17591171
负责人：
OKA Masahiro
金额：
$ 2.24万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

The signaling pathway of hepatocyte growth factor (HGF) through its receptor-tyrosine kinase c-Met was examined in human normal melanocytes and three malignant melanoma cell lines MeWo, HM3KO, and HMV-I. HGF-induced c-Met activation was observed in both melanocytes and melanoma cells, whereas phosphatidylinositol 3-kinase (PI3K), a downstream target of c-Met, was not activated in the melanocytes but enhanced in the melanoma cell lines. In contrast, extracellular signal-regulated kinase activity, another target of c-Met, was enhanced by HGF in both the melanocytes and melanoma cells. Therefore, the role of Gab1, the scaffolding adapter protein that couples activated c-Met and PI3K, was analyzed in these cells. Gab1 in the melanocytes showed an electrophoretic mobility slower than that in the melanoma cells, and the mobility shifted to that of the melanoma cells after treatment with alkaline phosphatase, indicating that Gab1 is highly phosphorylated on serine and threonine in the melanocytes. Introduction of protein kinase C (PKC) βII into the melanoma cells, which is expressed in melanocytes but absent in melanoma cells, using an adenovirus vector resulted in serine and threonine phosphorylation of Gab1 and also prevented tyrosine phosphorylation of Gab1 and its association with PI3K. Furthermore, the introduction of PKCβII suppressed HGF-induced activation of PI3K, and attenuated the in vitro invasion activity of the melanoma cells. These results indicate that the HGF signaling process from Gab1 to PI3K is negatively regulated by PKCβII, and that the loss of PKCβII is one of the steps for melanoma cells to gain invasive potential.

在人正常黑色素细胞和三个恶性黑色素瘤细胞系MEWO，HM3KO和HMV-I中检查了肝细胞生长因子（HGF）通过其受体 - 酪氨酸激酶C-MET的信号传导途径。在黑素细胞和黑色素瘤细胞中观察到HGF诱导的C-MET激活，而在黑素细胞中，磷脂酰肌醇3-激酶3-激酶（PI3K）是C-MET的下游靶标，但在黑素细胞中并未激活，但在黑色素瘤细胞系中增强了。相比之下，在黑色素细胞和黑色素瘤细胞中，HGF增强了细胞外信号调节的激酶活性，这是C-MET的另一个靶标。因此，在这些细胞中分析了伴侣激活C-MET和PI3K的脚手架衔接蛋白GAB1的作用。黑素细胞中的GAB1显示出比黑色素瘤细胞中的电泳迁移率慢，并且用醇磷酸酶处理后的迁移率转移到黑色素瘤细胞的迁移率转移到黑色素瘤细胞的迁移率，这表明GAB1在黑色素细胞中高度磷酸化在丝氨酸和三氨酸上。使用腺病毒载体将蛋白激酶C（PKC）βII引入黑色素瘤细胞中，但在黑色素瘤细胞中表达，但在黑色素瘤细胞中不存在，从而导致GAB1的丝氨酸和苏氨酸磷酸化及其与PI3K的关联。此外，PKCβII的引入抑制了HGF诱导的PI3K激活，并抑制了黑色素瘤细胞的体外侵袭活性。这些结果表明，从GAB1到PI3K的HGF信号传导过程受PKCβII负面调节，PKCβII的丧失是黑色素瘤细胞获得侵入性潜力的步骤之一。