Guo H B, Shen Z H, Huang C X, Ma J, Huang Y, Chen H L
Key Laboratory of Glycoconjugate Research, Ministry of Health, Shanghai Medical University, China.
Glycoconj J. 2000 May;17(5):315-22. doi: 10.1023/a:1007177806496.
The modulation of GnT-V activity by signaling molecules in PI-3-K/PKB pathway in human hepatocarcinoma cell line 7721 was studied. GnT-V activity was determined after the transfection of sense or antisense cDNA of PKB into the cells, as well as the addition of activators, specific inhibitors, and the antibodies to the enzyme assay system or culture medium. It was found that the basal activity of GnT-V was up regulated by the sense and down regulated by the antisense cDNA of PKB transfected into 7721 cells. GnT-V was activated by PIP2, PIP3 or GTPgamma[S] added to the assay system, and the activation of PIP2 or GTPgamma[S] was abolished by LY2940002, a specific inhibitor of PI-3-K, but the activation of PIP3 was not attenuated by LY2940002. In addition, GnT-V activity in cultured parental or H-ras transfected cells was inhibited by the antibody against PKB or PI-3-K. These findings demonstrated the involvement of PI-3-K/PKB signaling pathway in the regulation of GnT-V. Moreover, ET18-OCH3, an inhibitor of Raf translocation and PI-PLC enzyme, which produces the activator of PKC, as well as the antibodies against Raf-1 or MEK also inhibited GnT-V activity in the parental and H-ras transfected cells. The inhibitory rates, however, were less in the transfected cells than those in the parental cells. These results reveal that in parental and H-ras transfected 7721 cells, the basal activity of GnT-V is also regulated by the Ras/Raf-1/MEK/MAPK cascade in addition to PI-3-K/PKB signaling pathway. The significance of these two pathways in the regulation of GnT-V and their relations to the activation of PKC previously reported by our laboratory (Ju TZ et al., 1995 Glyconjugate J 12, 767-772) was discussed.
研究了人肝癌细胞系7721中PI-3-K/PKB途径中的信号分子对GnT-V活性的调节作用。将PKB的正义或反义cDNA转染到细胞中，并在酶分析系统或培养基中添加激活剂、特异性抑制剂和抗体后，测定GnT-V活性。结果发现，转染到7721细胞中的PKB正义cDNA上调了GnT-V的基础活性，而反义cDNA则下调了其活性。添加到分析系统中的PIP2、PIP3或GTPγ[S]可激活GnT-V，PI-3-K的特异性抑制剂LY2940002可消除PIP2或GTPγ[S]的激活作用，但对PIP3的激活作用无减弱效果。此外，抗PKB或PI-3-K的抗体可抑制培养的亲本细胞或H-ras转染细胞中的GnT-V活性。这些发现表明PI-3-K/PKB信号通路参与了GnT-V的调节。此外，Raf易位和PI-PLC酶的抑制剂ET18-OCH3（可产生PKC激活剂）以及抗Raf-1或MEK的抗体也可抑制亲本细胞和H-ras转染细胞中的GnT-V活性。然而，转染细胞中的抑制率低于亲本细胞。这些结果表明，在亲本细胞和H-ras转染的7721细胞中，除了PI-3-K/PKB信号通路外，GnT-V的基础活性还受Ras/Raf-1/MEK/MAPK级联反应的调节。讨论了这两条途径在GnT-V调节中的意义及其与本实验室先前报道的PKC激活的关系（Ju TZ等人，1995年，《糖缀合物杂志》12卷，767 - 772页）。
