BACKGROUND & AIMS:
:The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG-01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG-01 expressed mRNAs of PKC alpha, -beta I, -beta II, -delta, -epsilon, -eta, -theta and -zeta, but not PKC gamma. At the protein molecule level, MEG-01 was observed to express PKC alpha, -beta I, -beta II,- epsilon, -theta and -zeta, but lack -gamma, -delta and -eta. When differentiation of MEG-01 was induced by 100 nm 12-O-tetradecanoyl-phorbol-13-acetate (TPA), rapid translocation from cytosol to membrane fraction and down-regulation of PKC alpha, -epsilon and -theta was observed in 1-2h. On the other hand, PKC beta I and -beta II were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down-regulation, especially beta II, was very slow. The myristoylated, alanine-rich C kinase substrate (MARCKS) in the membrane fraction of MEG-01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time-course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKC alpha, -epsilon and -theta is involved in the initiation of differentiation, and that PKC beta I and -beta II have important roles in the maintenance of differentiation. Although PKC zeta was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.
背景与目标:
: 使用mRNA的热循环扩增和免疫印迹分析了不同蛋白激酶C (PKC) 同工酶在人巨核细胞白血病细胞系MEG-01的各种分化状态中的表达。MEG-01表达PKC α 、-β I、-β II、-δ 、-ε 、-eta、-θ 和-ζ 的mrna,但不表达PKC γ。在蛋白质分子水平上,观察到MEG-01表达PKC α,-β I,-β II,-ε,-θ 和-ζ,但缺少-γ,-δ 和-ζ。当100 nm 12 O-tetradecanoyl-phorbol-13-acetate (TPA) 诱导MEG-01分化时,在1-2小时内观察到从细胞质到膜级分的快速转运以及PKC α,-ε 和-θ 的下调。另一方面,观察到PKC beta I和-beta II不仅以不同的方式易位到膜部分,而且还易位到细胞骨架部分,并且它们的下调,尤其是beta II,非常缓慢。在整个分化过程中,观察到MEG-01细胞膜级分中的肉豆蔻基化,富含丙氨酸的C激酶底物 (MARCKS) 逐渐减少。此外,TPA处理的时程研究表明,将细胞孵育30分钟足以分化。这些结果强烈表明,PKC α,-ε 和-θ 的激活参与了分化的启动,并且PKC β I和-β II在维持分化中起重要作用。尽管PKC zeta对TPA处理具有抗性,并且其易位减少,但在整个分化过程中,胞质溶胶级分中该同工酶的数量减少了。