Role and regulation of phosphatidylinositol 3-kinase in melanoma cells

在人正常黑素细胞和三种恶性黑色素瘤细胞系MeWo、HM 3 KO和HMV-I中检测肝细胞生长因子（HGF）通过其受体酪氨酸激酶c-Met的信号传导途径。在黑素细胞和黑素瘤细胞中均观察到HGF诱导的c-Met激活，而c-Met的下游靶点磷脂酰肌醇3-激酶（PI 3 K）在黑素细胞中未激活，但在黑素瘤细胞系中增强。与此相反，细胞外信号调节激酶活性，另一个目标的c-Met，增强HGF在黑素细胞和黑色素瘤细胞。因此，在这些细胞中分析了Gab 1的作用，Gab 1是将活化的c-Met和PI 3 K偶联的支架衔接蛋白。Gab 1在黑素细胞中显示出比在黑色素瘤细胞中慢的电泳迁移率，并且迁移率在用碱性磷酸酶处理后转移到黑色素瘤细胞中，表明Gab 1在黑素细胞中的丝氨酸和苏氨酸上高度磷酸化。使用腺病毒载体将蛋白激酶C（PKC）βII导入黑色素瘤细胞（在黑色素细胞中表达，但在黑色素瘤细胞中不表达），导致Gab 1的丝氨酸和苏氨酸磷酸化，并阻止Gab 1的酪氨酸磷酸化及其与PI 3 K的结合。此外，PKCβII的导入抑制了HGF诱导的PI 3 K的激活，并减弱了黑色素瘤细胞的体外侵袭活性。这些结果表明，HGF信号通路从Gab 1到PI 3 K的过程受PKCβII负调控，PKCβII的缺失是黑色素瘤细胞获得侵袭潜能的步骤之一。

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.